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It was good to be back at Symbiosis. The festival has grown incredibly since the Eclipse at Pyramid Lake in 2012. It's now a world-class production, and the heads running the show deserve a lot of props. They've managed to capture the essence of community that makes Burning Man so special, but in their own authentic way. I can't wait to see where they take it next year for the Global Eclipse Gathering.
I was fortunate to be on the media team and learned a lot about shooting events. Shout out to Alexa Rae for giving me the chance to be on the team! There was so much to capture, with all the art, the stages, the beautiful people dressed in all the crazy outfits, and of course the scenery. After over 2000 exposures, and truthfully, I felt like I could have gotten a lot better shots. Crazy, huh?!
But that's what experience is all about. There are guys out there that do this for a living, and I respect them more than ever after this. You have to be on your A game and not sucked into the actual enjoying of the festival, and find the balance that works for you. I definitely made plenty of rookie mistakes, but being a photographer at the festival allowed me to connect with people in a new and more exciting way. In the end, I am just eternally grateful for the opportunity!
Enjoy the shots, there are quite a few... Bluebyrds and Sweaty Betty's were reppin' hard out there.


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Energizer maxi charger 2000 mAh.
Charges AA and AAA batteries.
Features 2 channels, timer shut off after 11 hours, Green LED to indicate charging status and polarity protection.
Includes 4 x AA 2000mAh batteries.
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This item takes 3-4 days to arrive instore. Grace the crowd in the halle gold and cream sequin formal dress from langhem. On trend cutaways falling into a sexy backless style this evening dress is the way to stand out at your next formal or red carpet event. In soft tones of cream and complemented by gold sequin the dress will have all eyes on you for any after dark affair. Features of the gown include a bodice that consists of a v neckline with bust cups that have been inserted into the evening dress keeping you comfortable and secure whilst wearing the dress. Falling to the floor in cream is a full length skirt this is a great style for your high school formal dress. Keeping you on trend is the cutaway bodice featuring symmetrical cutaways under the bust line which adds that sexy feel, with an exposed back not only keeps you on trend but adds that little bit of spice you are after whilst wearing this evening dress. Dress closure is with a centre back zip and hook and eye detailing you can easily step in and out of this gown. Model wears a size 10.


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A federal jury in San Francisco convicted Oakland "Guru of Ganja" Ed Rosenthal on Wednesday of three of the five marijuana-growing felonies of which he stood accused.
After starting deliberations Tuesday afternoon, jurors convicted Rosenthal, 62, of one conspiracy count; one count of growing, intending to distribute and distributing marijuana; and one count of using a commercial building — 1419 Mandela Parkway in Oakland — as a site for growing and distributing marijuana.
But they acquitted him of growing and distributing marijuana at the Harm Reduction Center medical-marijuana club on San Francisco's Sixth Street, and they deadlocked on whether he had conspired to do so.
U.S. District Judge Charles Breyer — who presided over Rosenthal's first trial in 2003 and has made it clear that he believes Rosenthal shouldn't have been retried — told Assistant U.S. Attorney George Bevan to call whichever superiors he needed for approval to have that final count dropped, as he wouldn't brook yet another retrial in his courtroom. The charge was dropped within about an hour.
So, more than six years after federal agents raided the Mandela Parkway warehouse, the Harm Reduction Center, Rosenthal's home and other sites, seizing thousands of marijuana plants, Rosenthal now faces no prison time, no fine and no probation at all.
That's because Bevan and Breyer agreed months ago that Rosenthal couldn't be sentenced now to anything beyond the one day of time, already served, to which he was sentenced for his 2003 convictions in the same case, overturned last year by the 9th U.S. Circuit Court of Appeals due to juror misconduct. Rosenthal is scheduled to be "sentenced" next Wednesday, but he'll walk free.
Prosecutors haven't publicly discussed their motives and goals in retrying Rosenthal. It could be that they wanted him to have felony convictions on his record should he ever be busted again, or to send a message to other medical marijuana advocates, or simply to chalk up a win in so long-running and high-profile a case.
"I think I'm going to flee to Canada, though, in the next 24 hours, just so they can bring me back for sentencing," Rosenthal quipped after Wednesday's verdict. "I feel like the whole thing is a parody. … It's not going to really change my life much one way or the other."
But he intends to appeal these convictions nonetheless as a travesty of justice, he said.
Robert Amparan, one of Rosenthal's attorneys, said he'll first file a motion for a new trial. Breyer seems unlikely to grant such a motion, given his disdain for this second trial. Amparan also said his own strength is jury trial, and he wants another attorney to review his work with fresh eyes, so he anticipates Rosenthal will retain new counsel for the appeal.
Prosecutors had reindicted Rosenthal in October with these charges as well as nine tax-evasion and money-laundering counts, but Breyer tossed out all the financial counts in March, deeming them vindictive prosecution.
As in his first trial, the jurors weren't allowed to hear any testimony that Rosenthal was acting under the auspices of the Oakland Cannabis Buyers Cooperative — deemed an officer of the city by Oakland's City Council — to grow marijuana for use under the state's medical marijuana law. Federal law still bans all cultivation, possession and use of marijuana.
"Whether they know it or not, the jury voted against their own self-interest," Rosenthal said. "At some point they're going to wake up and realize the enormity of what they did, and they're going to live with that for the rest of their lives the way the previous jury did."
Most of the jurors in Rosenthal's 2003 trial renounced their verdict within hours of rendering it, saying they felt the exclusion of his medical motives from the trial had railroaded them into convicting him.
"It's a cruel thing for the government to impose upon its citizens, the idea that they have to leave their conscience behind when they vote in the jury box. That should be part of it, and so should justice," Rosenthal said.
Amparan said he's concerned Rosenthal's reconviction will embolden federal authorities to crack down on medical marijuana throughout California and elsewhere. If the federal government could persuade a Bay Area jury to convict someone who'd been acting under Oakland's municipal authority, he said, everyone is now at risk "be they a dispensary, be they a grower, be they a patient."
Rosenthal said he's working with a pair of state legislators to draft a bill that would grant providers more explicit protection under state law; he expects to have an announcement on that within the next few weeks.


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Call (860) 646-6478 to get this deal
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|Actual Size:
|42" x 8' H
|Ideal for Ages:
|2 - 12 yrs
|Shipping Weight:
|75 lbs.
Triple Ball Toss for Commercial Playground Fun Hoop /w galvanized post
Like a playground version of basketball, this large basket adds a new element of fun to the classic ball toss. Kids just throw a ball into the top of the basket, and wait with anticipation and delight when the ball comes rolling out of one of the multiple exit routes. It's easier than basketball!
- Great for encouraging cooperation as well as social skills.
- Heavy-duty galvanized steel post withstands typical playground use and abuse.
- Wonderful for including children with special needs.
- Does not require significant investment in safety surfacing.
- Works on kids' coordination and muscle development while they have a blast!
- Available with powder coated post for $100 upcharge
- Also available without the post. Ask a sales rep for details.
Fully compliant with all public playground safety standards (ASTM & CPSC) and designed with commercial grade components for resisting corrosion, fading and mildew, this champion playground equipment has always been a bestseller! Available with red or green powder-coated post for a slight upcharge, choose your plastic color from several bright, inspiring colors as shown. Post measures 3.5 inch diameter, and the Toss Bucket measures 32" H, 42" opening and the three exit holes are 11" each. Buy it for your new playground project, or get it to spruce up the fun your existing play structures.


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The mission of the Statewide Independent Living Council of Hawai'i (SILC) is to promote independent living and the integration of persons with disabilities into the community and to aid individuals in achieving their goals and basic human rights.
Statewide Independent Living Council of Hawai'i (SILC):
Statewide Independent Living Council of Hawai'i (SILC) has council members that are appointed by the Governor. The majority of council members are persons with disabilities from communities statewide. The SILC is a not for profit, non-Governmental, consumer-controlled organization. The SILC works closely with Centers for Independent Living (CILs), the Designated State Entity (DSE) and consumers statewide to develop the three year State Plan for Independent Living (SPIL) as required by the Rehab Act of 1992, as amended. The council supports the independent living philosophy for everyone especially people with disabilities who expect equal access to employment, social, economic and political opportunities in their communities. The council also provides support and technical assistance to CILs in Hawai`i.
SILC Council Members
Jenny Hausler
Beverly Wong
Laura Tobosa
Judy Guajardo
Shannessy M. Ahu, Division of Vocational Rehabilitation (DVR)
Tammy Evrard, Ex-Official Non-Voting Member
Candice Carter
Roxanne Bolden, Executive Director, Aloha Independent Living Hawaii
Linda Lambrecht
Annette Tashiro
Sheryl Nelson, Executive Director
Barbara Burton, Administrative Assistant


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Eating and drinking are two most basic needs humans have. In order to satisfy this need, we are required to prepare food on a daily basis. The skills we used are not the same as the skills we use now. After all, we have frozen dinners and microwave ovens. But still, there are certain parts of food preparation we can't avoid. What's more, some of us really enjoy preparing food!
In this case, preparing food doesn't really depend on your skills only, the tools we use have an important role to play too. And one of more important tools in the kitchen are chef knife sets.
Every type of slicing, carving and cutting requires a different knife. Of course, you could use one knife, but it's much more easier to use specialized knives. The ones we usually use are Chef knife, paring knife, utility knife, carving knife, bread knife, cleaver and fillet knife. When you're out and about to buy a new chef knife set for yourself, make sure you check the blades. They can be either forged or stamped, and you should always go for the first kind as it has a better quality.
There are many chef knife sets to choose from and they are all made out with different materials, have different handles and use different types of blade. It's up to you to figure out what kid of set would be perfect for your needs and your budget. But, before you take price as a determining factor in buying a knife, think about these:
- What's your skill level?
- How often do you cook?
- What kind of meals you are going to prepare?
After you have the answers to these 3 questions, choose a suitable chef knife set for your kitchen. I'm sure you will find a perfect option quickly, as there are a lot of them on the market right now.


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You need to become a Contributor to join the discussion - Find out how.
Alex WeberHunter@lecanardmalade · Marketing & Design
I think it is a great idea and addresses a very important pain point for a lot of parents! Awesome job!
@lecanardmalade Thank you so much!!
Edward Shepard@edwardshepard
Fantastic! Every parent I know needs this.
@edwardshepard Thank you so much! We cannot wait to scale outside of Colorado.
8farmroad@8farmroad · Musician
Great idea...went through the whole registration process before they tell me it's only available in Boulder, CO :(
@adamrsweet You are right, we will update the messaging to make this more clear. Thank you for the feedback!
Uri Moszkowicz@apples_over_oranges · This is fun
Are there really 1500 camps in Boulder? I registered too, took me a while to figure out that it's only in Boulder. Wasn't really sure what to do here otherwise. Seems over engineered to me.
@apples_over_oranges There are over 1500 camps, classes and tutors geared for Boulder area parents (some are outside of the city). This is our launch and test market and we will be dialing in functionality and messaging so we can scale rapidly. Appreciate the feedback -- and updating geography messaging today.


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MeetUp - Yahoo Groups clone
Orçamento $100-300 USD
I need a script in php as a clone of Yahoo groups or Meet Up,
it shold include any features of these sites:
- Create and manage your own group
- Mailing List manager
- Read and reply to group messages. Start a new discussion.
- Share photos with group members
- Coordinate events for your group with a shared calendar
- Polls
I'm italian so i need a multilanguage script, i will do the italian translation from english
6 freelancers estão ofertando em média $253 para esse trabalho
Thanks for inviting us to place the bid. Offcourse we can do it eqasily as we have done some similar work in our past but the budget you mentioned is not good. You will have to pay atleast US$ 5000 for the exact featur Mais
To do this on your budget will be hard - as it requires direct tie in with a mail server... this is a quote to both host and to offer creation of groups for you... it won't have ALL the features of Yahoo Groups, but wi Mais
Hi, I can give u the exact clone. Please get in touch through Pmb
multilanguage (basetemplated) sql based data .. incl. backupfunctions, webadministration, documentation and installation


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What's the weather like where you are?
Become an ABC11 future meteorologist is as easy as downloading this form and recording the weather conditions where you live.
You can also download our rain gauge information and submit the results to us and receive your free ABC11 T-Shirt!
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Cytoplasmic filamentous rods and rings (RR) structures were identified using human autoantibodies as probes. In the present study, the formation of these conserved structures in mammalian cells and functions linked to these structures were examined.
Methodology/Principal Findings
Distinct cytoplasmic rods (∼3–10 µm in length) and rings (∼2–5 µm in diameter) in HEp-2 cells were initially observed in immunofluorescence using human autoantibodies. Co-localization studies revealed that, although RR had filament-like features, they were not enriched in actin, tubulin, or vimentin, and not associated with centrosomes or other known cytoplasmic structures. Further independent studies revealed that two key enzymes in the nucleotide synthetic pathway cytidine triphosphate synthase 1 (CTPS1) and inosine monophosphate dehydrogenase 2 (IMPDH2) were highly enriched in RR. CTPS1 enzyme inhibitors 6-diazo-5-oxo-L-norleucine and Acivicin as well as the IMPDH2 inhibitor Ribavirin exhibited dose-dependent induction of RR in >95% of cells in all cancer cell lines tested as well as mouse primary cells. RR formation by lower concentration of Ribavirin was enhanced in IMPDH2-knockdown HeLa cells whereas it was inhibited in GFP-IMPDH2 overexpressed HeLa cells. Interestingly, RR were detected readily in untreated mouse embryonic stem cells (>95%); upon retinoic acid differentiation, RR disassembled in these cells but reformed when treated with Acivicin.
RR formation represented response to disturbances in the CTP or GTP synthetic pathways in cancer cell lines and mouse primary cells and RR are the convergence physical structures in these pathways. The availability of specific markers for these conserved structures and the ability to induce formation in vitro will allow further investigations in structure and function of RR in many biological systems in health and diseases.
Citation: Carcamo WC, Satoh M, Kasahara H, Terada N, Hamazaki T, Chan JYF, et al. (2011) Induction of Cytoplasmic Rods and Rings Structures by Inhibition of the CTP and GTP Synthetic Pathway in Mammalian Cells. PLoS ONE 6(12): e29690. https://doi.org/10.1371/journal.pone.0029690
Editor: Wael El-Rifai, Vanderbilt University Medical Center, United States of America
Received: June 9, 2011; Accepted: December 2, 2011; Published: December 29, 2011
Copyright: © 2011 Carcamo 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 in part by a grant from the National Institutes of Health grant Al47859. WCC is supported by Bridges to Doctorate, University of Florida Graduate School, and University of Florida Alumni Graduate Fellowship. ST is supported by the University Scholars Program. Publication of this article was funded in part by the University of Florida Open-Access Publishing Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study.
Competing interests: The authors have declared that no competing interests exist.
Over the past few decades many human autoantibodies have emerged as significant disease-specific markers for systemic rheumatic diseases [1]. These autoantibodies are mainly directed against intracellular macromolecular complexes or particles, such as nucleosomes and small nuclear/cytoplasmic ribonucleoproteins [1]. Thus, human autoantibodies have also served as useful probes for exploring subcellular structures and functions because of their unexpected specificity to novel self-antigens. Examples of significant uses of human autoantibodies in further characterization of novel subcellular structures included the identification of p80-coilin in Cajal bodies (formerly known as coiled bodies) [2] and GW182 in GW bodies [3]. Within the past few years, our laboratories identified novel human autoantibodies that recognized unique cytoplasmic structures described provisionally as rods and rings. The current study reports the identification of CTPS1 and IMPDH2 as components associated with these mammalian RR.
CTPS1 and IMPDH2 are key enzymes in the biosynthetic pathway for cytidine triphosphate (CTP) and guanosine triphosphate (GTP), respectively. CTPS1 catalyzes the rate-limiting step in generating CTP from uridine triphosphate. CTP is involved in nucleic acid and phospholipid biosynthesis and plays an important role in controlling cellular proliferation [4]. Two isoforms, CTPS1 and CTPS2, have been identified with 74% amino acid similarity. The 67 kDa CTPS1 is a target for antiviral, antineoplastic, and antiparasitic drug development. Currently available specific inhibitors of CTPS1 include Acivicin and 6-diazo-5-oxo-L-norleucine (DON) [5].
The oxidation of inosine monophosphate to xanthosine monophosphate is the rate-limiting step in the de novo guanine synthetic pathway catalyzed by IMPDH2, which is known to have a role in regulating cell proliferation. Due to its rate-limiting property, it has been a major target for immunosuppressive, antiviral, and cancer chemotherapy. Two isoforms of IMPDH, IMPDH1 and IMPDH2, sharing 84.0% sequence identity and 95.3% similarity have been identified and both are 56–58 kDa proteins that function as tetramers [6]. A study on the regulation of IMPDH with mycophenolic acid (MPA) showed that MPA binds to IMPDH and causes a conformational change resulting in the formation of inactive angular aggregates [7]. IMPDH2 inhibitors such as MPA, Ribavirin, and Tiazofurin are currently used for various medical conditions [8]–[11].
In the current report, inhibiting either CTPS1 or IMPDH2 using different compounds demonstrated induction of RR in a variety of cell types. More importantly, RR were identified in mouse embryonic stem cells (ESCs) and their disassembly was observed upon induced differentiation.
Rods and rings in the cytoplasm
A distinct cytoplasmic pattern was first identified in a routine antinuclear antibody (ANA) test using HEp-2 cell slides from INOVA Diagnostics Inc. These ANA slides are generally used in clinical laboratories for patients developing antibodies to self-proteins/antigens as a diagnostic screening assay for autoimmune diseases. The structures recognized by a prototype human serum 604 were distinct cytoplasmic rods (∼3–10 µm in length) and rings (∼2–5 µm in diameter, Figure 1). On average, there are one to two rods and/or rings per cell including some interesting apparent intermediate structures such as a figure "8", an elongated ring, twisted ring, rods with pin loops at one end, as well as some that appeared to be transitioning from rods to this peculiar form (Figure 2). Immunofluorescence was performed to visualize the RR structure and location with respect to the nucleus and the Golgi complex (Figure 1A). Some rods often align adjacent to the nucleus (short arrows, ∼40% frequency) or perpendicular to the nucleus (long arrows, ∼40% frequency), and rings may be found in the cytoplasm (arrowheads, 5–10% frequency). RR are expressed in some mitotic cells; however, co-staining studies with anti-centromere protein F (CENP-F), showed that the expression of either rods or rings were independent of the cell cycle, as they appear in all stages (Figure 3). CENP-F is a cell cycle marker that has little or no expression at the G1 phase, with increasing level during the S phase, and reaching highest expression during late S and G2 phases [12], [13].
(A) Merged image of HEp-2 co-stained with human anti-RR prototype serum 604/Alexa 488 goat anti-human Ig (green) and rabbit anti-giantin (Golgi marker)/Alexa 568 goat anti-rabbit Ig (red). Rods are often presented adjacent (short arrows) or perpendicular (long arrows) to the nucleus while rings (arrowheads) are found either 1 or 2 to a cell. M, mitotic cell. HEp-2 cells were also co-stained with serum 604/Alexa 488 goat anti-human Ig (green, B,E,H) and different cytoplasmic markers using mouse anti-tubulin (C), anti-vimentin (F), anti-pericentrin (I), followed by Alexa 568 goat anti-mouse Ig (red). Nuclei were counterstained with DAPI (blue). Bar, 10 µm.
(A) Figure "8" structure (arrowhead) with a curved rod adjacent to the nuclear envelope; (B) elongated ring; (C) elongated, twisted ring (short arrow); (D) rod with a pin-loop (long arrow); (E) Figure "8" structure (arrowhead); (F) another rod with pin-loop (long arrow). Nuclei were counterstained with DAPI (blue). Bar, 5 µm.
HEp-2 cells were co-stained with rabbit anti-CENP-F/Alexa 568 goat anti-rabbit IgG (A) and human anti-RR serum 604/Alexa 488 goat anti-human IgG (B). G1 cells had little or no CENP-F staining, whereas late S/G2 and mitotic (M) cells showed strong staining for CENP-F. Nuclei counterstained with DAPI (blue). Bar, 10 µm.
To examine the relationship of RR with other known cytoplasmic organelles, co-localization studies were performed using known markers for cytoplasmic structures. Co-staining studies with antibodies to the Golgi complex showed that RR were independent of the Golgi complex (Figure 1A). Although many rods appeared to be in the same vicinity as the Golgi complex, there are also many clear-cut examples of rods unassociated with this subcellular structure. Cytoplasmic structures, referred to as "actin rockets," were previously described in bacteria-infected cells [14]. To determine whether RR are related to actin rockets, co-staining with mouse anti-actin antibody was performed to show that RR are not enriched in actin (data not shown). Furthermore, upon careful examination at higher magnification, the HEp-2 cells in our laboratory were not contaminated with bacteria and cell viability was >99%. Since RR are filamentous, co-staining experiments were also performed to determine whether tubulin or vimentin, was enriched in RR. Costaining showed that RR are not enriched in tubulin (Figure 1B–D). Co-staining of human prototype serum 604 and anti-vimentin antibody ruled out enrichment of RR with vimentin (Figure 1E–G).
Another initial consideration was that primary cilia, which function as nonmotile sensory organelles and have a rod-like appearance [15], could be a possible candidate. To rule out RR as primary cilia, a co-staining study with anti-pericentrin, which recognizes centrosomes, was conducted. Centrosomes are the precursors of primary cilia and are often located at the ends of the primary cilia. Since centrosomes were not located at the ends of rods recognized by human prototype serum 604 (Figure 1H–J), these rods are not primary cilia. In fact, there is no noticeable association of either rods or rings with centrosomes. Furthermore, upon closer examination, the fact that ring-type structures have never been observed for primary cilia is consistent with the conclusion.
These findings are highly reproducible using HEp-2 cell substrate from INOVA, as well as home-made cell slides prepared with inhibitor-treated cells (see below) using various commonly used fixatives in biological laboratories including acetone at −20°C for 5 minutes, acetone/methanol (1∶1) for 10 minutes, or 3% paraformaldehyde and 0.1% triton-X. Co-staining experiments with known subcellular markers showed no association with known cytoplasmic organelles such as GW bodies (data not shown).
Identification of CTPS1 and IMPDH2 as RR components
During the initial phase of our study, CTPS1 became a candidate RR protein because, in the Carnegie protein trap study, it was noted that similar rod-like structures were observed in Drosophila embryos carrying GFP-CTPS1 [16], [17]. Co-staining studies with rabbit anti-Drosophila CTPS antiserum showed localization of CTPS1 to RR, identified with prototype human anti-RR serum It2006 (Figure 4A). As there are 75.0% sequence identity and 89.8% similarity between human CTPS1 and CTPS2, antiserum to CTPS1 probably crossreacts with CTPS2 and both might be components of RR.
(A) Enrichment of CTPS1, detected by rabbit anti-CTPS1 (green), to RR (arrows) identified by It2006 (red). Nuclei were counterstained with DAPI (blue). (B) IMPDH2 stained by rabbit anti-IMPDH2 (red) localized to RR (arrows) detected by It2006 (green). Bar, 10 µm. (C) Immunoprecipitation (IP) analysis using an extract of [35S]-methionine-labeled K562 cells and rabbit anti-IMPDH2, It2006, and a second human anti-RR serum 609. It2006 recognized a 55 kDa protein band that co-migrated with IMPDH2 immunoprecipitated by rabbit anti-IMPDH2. Serum 609 did not immunoprecipitate the 55 kDa protein. (D) IP-Western blot demonstrated that serum It2006 immunoprecipitated IMPDH2, which was recognized by both mouse monoclonal and rabbit anti-IMPDH2 antibodies. NHS, control normal human serum.
A second candidate RR protein was postulated in part because of an isolated ring-like structure in the cytoplasm identified by a single autoimmune serum recognizing the enzyme IMPDH2 [18] and the consideration that CTPS1 and IMPDH2 are enzymes in closely linked pathways as discussed below. In the first experiment to demonstrate whether IMPDH2 was localized to RR, a commercially available rabbit anti-IMPDH2 antibody was used to co-stain HEp-2 cells. Anti-IMPDH2 stained both rods and rings (Figure 4B, arrows). Immunoprecipitation analysis using an extract of [35S]-methionine-labeled K562 cells showed that rabbit anti-IMPDH2 and It2006 both pulled down a 55 kDa protein (Figure 4C). Furthermore, IP-Western analysis confirmed that the 55 kDa protein immunoprecipitated by It2006 was IMPDH2, since it was recognized by both mouse monoclonal anti-IMPDH2 and rabbit anti-IMPDH2 (Figure 4D). Since human IMPDH1 and IMPDH2 have 95.3% sequence similarity, our data cannot exclude IMPDH1 is also enriched in RR.
Expression of rods and rings in cultured cells
HEp-2 cell slides from INOVA Diagnostics have been consistently positive for RR, tested from lot to lot, since this study was initiated over five years ago. The same was not true for HEp-2 cell ANA slides from other manufacturers examined to date, with staining varying from completely negative to granular or diffused cytoplasmic staining. This could be the explanation for why the subcellular staining observed by Blüthner et al [18] was largely different from ours, except for showing one or two isolated ring structures. These differences among products from several manufacturers remains a question not completely resolved, but may stem from differences in culture conditions, proprietary sample processing, or both. Homegrown HEp-2 cells on coverslips or 8-chambered slides have generally not been positive for RR staining unless cells were kept in rapidly dividing log phase or under subconfluent cell density. Confluent cultures were generally negative for RR. Variability from experiment to experiment was clearly observed, and on average only 5–30% of cells were positive for RR detectable by serum 604 or It2006. Cells were always co-stained with both human anti-RR and rabbit anti-giantin (Golgi marker) antibodies to ensure that variables were not introduced in the fixation and immunostaining steps in these experiments. Exactly how INOVA Diagnostics can produce highly consistent HEp-2 cell slides with >95% cells positive for RR staining remains unresolved, but clearly the difference must stem from their unique manufacturing methods. Thus, HEp-2 cells do not express RR under our standard culture conditions. Cells that do not ordinarily express RR have a diffused cytoplasmic pattern when stained with prototype human serum (Figure 5A). However, it was noted that cells recently thawed from liquid nitrogen express RR in about 15% of the cells. Treating cultured cells with 0.015% to 0.5% of DMSO overnight to simulate freezing medium condition (often with 10% DMSO) did not induce formation of RR.
Untreated HEp-2 cells (A) and cells treated with inhibitors 2 mM DON (CTPS inhibitor, B), 2 mM Acivicin (CTPS inhibitor, C), or 2 mM Ribavirin (IMPDH2 inhibitor, D) for 24 h were co-stained with human anti-RR serum It2006 (green) and rabbit anti-giantin (red). Nuclei were counterstained with DAPI (blue). The percentage of cells with RR displayed for each experiment is shown in the lower right corner with the total number of cells counted indicated in parentheses. Bar, 10 µm.
Induction of rods and rings in cultured HEp-2 cells
When CTPS1 and IMPDH2 were identified as RR components, the next series of experiments focused on the effects of inhibiting these enzymes. Effects of each drug treatment were analyzed by immunofluorescence (Figure 5). The first applied was the CTPS1 inhibitor, DON, on HEp-2 cells cultured at 2 mM, a functional concentration reported in literature [5], [19]. A surprising finding was that both rods and rings were readily detected in 96.7% of cells after overnight incubation (Figure 5B) compared to 0% in untreated control cells cultured in parallel (Figure 5A). The RR detected in DON-treated cells were identical in frequency to those observed in the INOVA HEp-2 slides. A second independent CTPS1 inhibitor Acivicin [4], [5], as well as the IMPDH2 inhibitor Ribavirin, also showed the ability to induce RR formation under similar conditions (Figure 5C and D). In addition, RR induced by 2 mM DON, known to inhibit CTPS1 but not IMPDH2, were detectable with the anti-IMPDH2 antibody along with the human anti-RR serum. Therefore, CTPS1 and IMPDH2 appeared to aggregate after induction with either inhibitor to form RR.
Inhibition of CTPS1 or IMPDH2 induced RR formation in a concentration-dependent manner (Table 1). The three inhibitors had different efficiency in the induction of RR formation in HEp-2 cells. Ribavirin was the most potent inducer of RR; even at concentrations lower than 7.81 µM, Ribavirin induced RR in nearly all cells, whereas DON and Acivicin did not induce RR efficiently at these low concentrations (Table 1, p<0.0001). Significant induction of RR by Ribavirin vs DON was observed at a concentration lower than 0.5 mM (p<0.0001) and vs Acivicin at a concentration lower than 15.6 µM (p<0.0001). Induction of RR by Acivicin vs DON was significant between 0.25 mM and 15.6 µM (p<0.0001).
There are also differential time requirements for RR induction in HEp-2 cells treated with inhibitors at a fixed 2 mM concentration (Table 2). Ribavirin induced RR as early as 30 min and between 1 and 24 h, nearly all cells had RR. Induction of RR by Ribavirin was significantly faster than DON, which in turn was significantly faster than Acivicin (Table 2).
On average, cells treated with DON or Acivicin had two RR per cell (Figure 5B, 5C), while cells treated with Ribavirin had more than two RR per cell (Figure 5D). The additive effect of two inhibitors on RR formation was also investigated. Addition of both DON and Ribavirin at 2 mM did not have a noticeable effect on RR formation since both alone induced formation of RR in >95% of treated cells. However, when inhibitors were applied at lower concentrations and for shorter periods, obvious additive effects were observed. For example, treatment of HEp-2 cells with 1 mM DON for 3 h induced RR in 52% of cells; however, when this was combined with 1 mM Ribavirin for 30 min, which alone induced RR in 13% of cells, RR was detected in 91% of cells. In addition, when HEp-2 cells were exposed to lower concentrations of Ribavirin (2 µM, 3 h), the RR structures were small and difficult to visualize; however when combined with DON (1 mM, 3 h), typical RR structures were observed. Thus, inhibition of both CTPS1 and IMDPH2 had apparent additive effects on RR formation, supporting the finding that they aggregated in the same structures. An unrelated compound, enoxacin, used as an oral broad-spectrum fluoroquinolone antibacterial agent observed to enhance miRNA activity [20] served as an additional negative control as it did not induce RR after 24 h treatment in various concentrations (10–100 nM). Thus, the inhibition of either CTPS1 or IMPDH2 appeared to be specific for the induction of RR in HEp-2 cells.
Induction of RR in other cancer and primary cells
To address whether the induction of RR is conserved, multiple cell lines were analyzed, including other adherent monolayer cell lines like HeLa (Figure 6A), CAL 27 (Figure 6B), and HCT116 (Figure 6D), as well as a suspension monocytic cell line THP-1 (Figure 6C). Cells were incubated with DON for 24 h and co-stained with serum It2006 and anti-giantin. All cell lines expressed RR at a high frequency (∼98% for HeLa, CAL 27, and THP-1), comparable to the frequency observed in HEp-2 cells except HCT116 cells, with only 35.8% of cells positive for RR. Untreated cells normally did not show expression of RR, but isolated HeLa cells showed some cytoplasmic granular staining and THP-1 produced some short "immature" rods. To determine whether the expression of RR could be observed in species other than human, mouse 3T3 fibroblasts and rat NRK cells were stained with It2006. Freshly defrosted 3T3 and NRK often showed expression of RR (5 to 25% of cells) in the first one or two passages, but their expression disappeared completely in subsequent passages. The ability of freshly thawed cells to express RR was not examined further due to the lack of consistency between batches of frozen cells. Mouse primary cardiomyocytes, as well as the often co-purified endothelial cells and fibroblasts isolated from neonatal heart, did not express RR until induced with 2 mM DON in overnight culture (Figure 7). Overall 88.4% of cells had detectable RR, but cardiomyocytes detected by co-staining of actinin had up to 92.6% showing induced RR (Figure 7B). Thus, RR expression appeared to be conserved as they could be induced in multiple immortalized cells, as well as in primary cultured cells.
Induction of RR observed in human cancer cell lines HeLa (A), CAL 27 (B), THP-1 (C), and HCT116 (D) when treated with 2 mM DON in culture for 24 h, fixed, and co-stained with human anti-RR serum It2006 (green) and rabbit anti-giantin (red). Untreated controls showed few or no RR. The percentage of cells with RR displayed for each condition is shown in the upper right corner with the total number of cells counted indicated in parentheses. Bar, 5 µm.
Mouse primary cardiomyocytes prepared together with fibroblasts and endothelial cells, were treated with 2 mM DON and cultured for 24 h. Cells were co-stained with human anti-RR serum IT2006 (green) and mouse anti-actinin monoclonal antibody (red). Nuclei counterstained with DAPI (blue). Actinin-positive cardiomyocytes (A, B, red) as well as actinin-negative fibroblast or endothelial cells (A, C) all show distinct rods. The percentage of cells with RR displayed is shown in the lower right corner with the total number of cells counted indicated in parentheses (A, all cells; B, actinin-positive cardiomyocytes only; C; actinin-negative fibroblast and endothelial cells). Bar, 10 µm.
Induction of RR is quantitatively sensitive to the level of cellular IMPDH2 protein
To monitor the formation of RR, the use of GFP fusion constructs was explored. HeLa cells were transfected with GFP-IMPDH2 (Figure 8A–C) or the GFP vector alone (Figure 8D–F). Following transfection, cells were treated with 2 mM of Ribavirin for 24 h, and then fixed and stained with rabbit anti-IMPDH2. GFP-IMPDH2 transfected cells had brighter fluorescence compared to untransfected cells, confirming the expression of recombinant IMPDH2 (Figure 8A). Surprisingly, GFP-IMPDH2 transfected cells did not show RR after Ribavirin induction, whereas neighboring untransfected cells showed RR as expected (Figure 8C). To rule out transfection-induced artifacts interfering with the induction of RR, the induction of RR in control GFP-vector transfected cells was not affected by the transfection (Figure 8F). Series of experiments were performed to explore whether the lowest expression of transfected GFP-IMPDH2 could produce cells with GFP-labeled RR by titrating the amount of transfected plasmid and by reducing the time of transfection. None of these experiments yielded live cells labeled with GFP-RR. Therefore, the overexpression of IMPDH2 appeared to inhibit formation of RR, even at high concentrations of Ribavirin. When these GFP-IMPDH2 transfected cells were exposed to higher concentrations of Ribavirin up to 8 mM, only small aggregates were observed in some of the transfected cells.
HeLa cells were transfected with either GFP-IMPDH2 (A–C) or a GFP vector (D–F) for 24 h and followed by 2 mM Ribavirin incubation for 24 h. Cells were stained with rabbit anti-IMPDH2 (red) and counterstained with DAPI (Blue). GFP-IMPDH2 transfected cells (T) did not express RR while untransfected cells showed rods (arrows) and rings (arrowheads). RR were detected in both GFP vector transfected and untransfected cells. Bar, 10 µm.
Since overexpression of IMPDH2 inhibited RR induction, the next series of experiments were to address whether knockdown of CTPS1 or IMPDH2 would affect the induction of RR. The adopted strategy was to examine cells transfected with either CTPS1 siRNA (siCTPS1) or IMPDH2 siRNA (siIMPDH2). Thus, HeLa cells were either untransfected or transfected with siCTPS1 or siIMPDH2 for 44 h, followed by an additional 4 h of incubation with three different concentrations of Ribavirin at 2 mM, 0.2 µM, or 0.05 µM. The 2 mM Ribavirin concentration was selected as the highest concentration tested and 0.2 µM was selected as the lowest based on the data presented in Table 1. Not shown in Table 1 for HEp-2 cells, 0.05 µM Ribavirin was tested in HeLa cells and was shown to be ineffective in the induction of RR. The induction of RR was monitored by double immunofluorescence using rabbit anti-IMPDH2 antibody as well as It2006. After siIMPDH2 knockdown, Western blot analysis showed reduction of IMPDH2 levels to 5–9% in untreated cells, siCTPS1 had little or no effect on the level of IMPDH2 as expected, although siCTPS1 was confirmed by real-time PCR to be functional with 62% CTPS1 mRNA knockdown (Figure 9A). The number of cells with RR were counted and plotted as a percentage (Figure 9B). While siCTPS1 transfection did not affect the percentage of cells positive for RR in all three concentrations of Ribavirin examined, knockdown of IMPDH2 showed significant increase in the percentage of cells with detectable RR formation at both 0.2 µM and 0.05 µM of Ribavirin (p = 0.0001 for 0.2 µM and 0.05 µM compared to untransfected and siCTPS1 transfected cells by Fisher's exact test). For the 2 mM Ribavirin treatment, although RR were induced in 100% of cells transfected with siIMPDH or siCTPS1, knockdown of IMPDH2 showed a decrease in the size of RR (with the average length of rod at 4.6 µm in siIMPDH2 transfected versus 6.9 µm in untransfected) along with the number of RR per cell (4.5 RR/cell in siIMPDH2 transfected versus 7.1 RR/cell in untransfected, p = 0.0024 by t-test) when compared to untransfected cells at 2 mM Ribavirin. At 0.2 µM Ribavirin, increased formation of RR was observed in cells with reduced levels of IMPDH2 (42.9% of cells in siIMPDH2 transfected vs 21.6% for siCTPS1, p = 0.0048). At 0.05 µM Ribavirin, siIMPDH2 transfected cells had 22.4% cells with shorter rods (Figure 9D) compared to siCTPS1 transfected cells with 3.6% (Figure 9C, p = 0.0001). Thus, the data show that the formation of RR by Ribavirin was sensitive to the total IMPDH2 level and excess intracellular IMPDH2 made cells resistant to the formation of RR. Therefore, RR formation appeared to depend on the molecular ratio of Ribavirin to IMPDH2 and cells with a lowered IMPDH2 level could be induced to form RR at low concentration of Ribavirin.
HeLa cells were either untransfected, transfected with siCTPS1, or siIMPDH2 for 44 h and followed by incubation of Ribavirin at various concentrations for an additional 4 h. (A) IMPDH2 expression was monitored by Western blot using rabbit anti-IMPDH2 antibody. Serial dilutions of untreated HeLa cell lysates (100%, 50%, and 25%) were used to help to comparatively quantitate the degree of protein knockdown. The level of actin was used as a loading control. Relative IMPDH2 protein levels were quantified using image J and normalized to the serial dilution standards. (B) Percentage of cells with RR were quantified using Mayachitra Imago with a range of 131 to 298 cells counted per data point. Cells treated with siIMPDH2 had a significant increase in percentage of cells with RR at 0.2 µM and 0.05 µM of Ribavirin analyzed by Fisher's exact test. (C–D) Representative image of cells at the lowest concentration of Ribavirin transfected with siCTPS1 or siIMPDH2. Arrows, rods; arrowhead, ring. Bar, 5 µm.
RR expression in embryonic stem cells
From the data presented in the previous sections, except for the unique case of the INOVA HEp-2 cells, various cultured cell types did not consistently express RR unless induced with one of the CTPS1/IMPDH2 inhibitors. The next question was whether certain cell types were uniquely capable of expressing RR in the absence of inducers. ESCs are highly proliferative cells that can self-renew and are pluripotent. Undifferentiated, uninduced ESCs were found to express RR (Figure 10B) similar to those described in HEp-2 cells (Figure 1A) with 1 or 2 RR per cell. Interestingly, unlike HEp-2 cells or mouse 3T3 cells (Figure 10A), where only ∼10% of cells had rings, >90% of ESC expressed rings, and <5% of cells had rods (Figure 10B). In contrast, ESCs that were differentiated with retinoic acid (RA) for 4 days no longer expressed RR (Figure 10C), but those differentiated ESCs remained capable of RR formation upon treatment of 2 mM Acivicin for 24 h and the RR phenotype was similar to the original undifferentiated ESC (Figure 10D). RR expression could be induced in multiple cancerous and primary cells, but RR were found to be expressed in untreated stem cells.
(A) Mouse 3T3 cells for comparison shown with both rods (80–90%, arrowhead) and rings (10–20%, arrows). (B) Undifferentiated mouse ESCs shown with ∼90% rings (arrows) and few rods. (C) ESCs treated with RA for 4 days showed no RR. (D) RR were induced in these RA-differentiated cells by treatment with 2 mM Acivicin for 24 h. Cells were stained with human anti-RR serum It2006 (green, A–D) and co-stained with rabbit anti-giantin (red, A, B only). Nuclei counterstained with DAPI (blue). The percentage of ESCs with RR displayed for each condition is shown in the lower right corner with the total number of cells counted indicated in parentheses. Bar, 5 µm.
In this study, a novel cytoplasmic structure was identified by human autoantibodies produced by patients with chronic hepatitis C virus (HCV) infection through ANA testing. Our preliminary data showed that only a subset of HCV patients had anti-RR antibodies. Only ∼25% of HCV patients treated with both interferon-α and Ribavirin, the most common combination therapy for HCV infection, had anti-RR antibodies while none of the untreated HCV patients developed an anti-RR response. Based on our present data that primary cells exposed to Ribavirin could form RR in vitro, one can speculate that cells from patients exposed to Ribavirin will form RR. Aggregated/modified IMPDH (and CTPS) in RR in these Ribavirin treated patients may become antigenic and trigger an autoimmune response.
Most subcellular organelles disassemble prior to mitosis and reform after cell division. Although some mitotic cells were devoid of RR and thus suggesting RR might disassemble prior to mitosis, either the disassembly of RR was incomplete or the disassembly did not occur at all in many cells, since ∼50% of mitotic cells, including all stages, clearly retained RR (data not shown). At least one autoantigen, IMPDH2, recognized by the human anti-RR prototype serum It2006 was determined through IP-Western analysis. Co-localization studies determined CTPS1 to be a second component of RR. Inhibitors of the two enzymes induced formation of RR in cells that did not express RR during continuous culture. RR induction could be accomplished with low nanomolar inhibitor concentrations. Overexpression of IMPDH2 inhibited formation of RR while knockdown of IMPDH2 protein allowed for more sensitive induction of RR with ultralow concentrations of Ribavirin that normally did not induce RR. Furthermore, in HeLa cells overexpressing IMPDH2, higher concentrations of Ribavirin were not effective and could only induce remnant of RR. Therefore, the induction of RR is inhibitor-concentration dependent with lower levels of IMPDH2 favoring RR formation in the presence of low level of Ribavirin.
Since CTPS1 and IMPDH2 are major enzymes controlling the biosynthetic pathways for CTP and GTP, respectively, the formation of RR using multiple independent inhibitors of these enzymes implies that this cytoplasmic structure is linked to these cellular metabolic pathways. It is, however, acknowledged that DON and Acivicin are not completely specific for CTPS1 but are not expected to affect activity of IMPDH [21]. The observations that the formation of RR is sensitive to the concentration of IMPDH2 (Figure 9) and that RR are present in some cases in untreated cells suggest that the formation of RR is linked to the functional level of these enzymes in cellular regulation related to ribonucleotide (CTP or GTP) synthesis. Since the inhibition of CTPS1 causes formation of RR that can be detected using an IMPDH2 antibody, CTPS1 and IMPDH2 must aggregate together to form RR, although demonstration of their direct molecular interaction is lacking. Due to the large size of these structures, one may speculate that there are many other components involved in RR formation and some of these may serve as molecular linkers to facilitate interaction between CTPS and IMPDH. Future biochemical analyses are needed to explore whether there is direct interaction of CTPS and IMPDH.
RR-like structures are evolutionarily conserved
Since our initial observation of RR over four years ago, morphologically similar structures were identified in five recent reports [17], [22]–[25] in addition to two previous reports [7], [26]. The first report of RR-like structures may be the 1987 study by Willingham et al. [26]. They reported a cytoplasmic "fibrillar structure" identified by an IgG3 monoclonal antibody generated from a Balb/c mouse immunized with mouse tumor cells. These structures were identified using transmission electron microscopy and immuno-electron microscopy as "large single paracrystalline arrays of individual filaments" not enclosed by a lipid bilayer membrane. Their structures were similar in appearance and number per cell to the RR in this study. The target recognized by the monoclonal antibody, an unknown 58 kDa protein in immunoprecipitation of [35S]-methionine radiolabeled cell lysate, was referred to as nematin (from nematodes). The identity of the antigen was not elucidated [26]. Similar to our results, they reported that these structures were unrelated to tubulin or vimentin and evolutionarily conserved in human, mouse, and rat. It was also reported that the nematin structure occurred following thawing of stored frozen cells [26] such as HEp-2, 3T3, and NRK cells, also observed in our laboratory.
The second study reporting RR-like structures was by Ji et al. identifying IMPDH2 protein aggregates formed from inhibition of enzymatic activity through MPA treatment [7]. The structures were identified by immunofluorescence and electron microscopy analysis [26]. Further analyses revealed that the aggregates were reversible with the addition of guanosine and its derivatives. The structures in that study and in ours were induced with two independent IMPDH2 inhibitors, MPA versus Ribavirin. Both inducers inhibit IMPDH2, but MPA is a noncompetitive and reversible inhibitor [27], while Ribavirin is a nonreversible inhibitor.
Ingerson-Mahar et al. demonstrated that CTPS formed filaments in C. crescentus [23]. Identification of the structure was through electron cryotomography along the inner curvature of C. crescentus. The metabolic enzyme CTPS has a bifunctional role, as a filament that regulates curvature and performs its catalytic function. A key difference was that treatment of DON in this study dissociated the CTPS filaments in bacteria, while DON induced the formation of RR in mammalian cells in our study. The latter finding on the effect of DON on mammalian cells is completely consistent with data in a recent report by Chen et al [25].
Other recent reports identified RR-like structures in both yeast and Drosophila. Noree et al. identified four novel filaments that were identified by screening a yeast GFP strain collection [24]. Through inhibition of CTPS, they determined that filaments were comprised of inhibited CTPS, similar to our results. In addition, they were able to visualize CTPS filaments in the Drosophila egg chamber, a subset of gut cells and neurons. Another report also identified an intracellular structure containing CTPS in Drosophila cells [17]. The structures, referred to as cytoophidia, were identified in ovary cells and other tissues [25], [28]. GFP protein trap fly stocks from the Carnegie Protein Trap Library identified CTPS as the component of cytoophidia. RR and cytoophidia may be the same conserved structures among different species [25].
Ramer et al. identified a large intracellular neuronal organelle that occurs in a subset of adult rat sympathetic ganglion neurons [22]. The structure, identified by immunohistochemistry plus confocal and electron microscopy, occurred mainly in a toroidal shape, but also as twists or rods. The structures, referred to as loukoumasomes or donut-like structures, were recognized by a monoclonal antibody that was raised against neuron-specific βIII-tubulin, but not recognized by other βIII-tubulin antibodies. One difference between this and our study was the increased toroidal (ring) structures in neurons, while we observed more rods than rings in all induced cancer cells, but more rings than rods in ESCs.
Spontaneous RR versus inhibitor-induced RR
Although RR may appear to be highly conserved as discussed above, our preliminary screening in mouse liver, kidney, and spleen using indirect immunofluorescence did not detect these structures. Undifferentiated ESCs express RR without the use of inhibitors while differentiated cells do not express RR and, interestingly, differentiated cells were still capable of RR formation upon induction with a CTPS1 inhibitor. However, this should not be interpreted as de-differentiation back to stem cells. A note of caution is needed, as it is not clear whether ESCs RR are structurally and functionally the same as those of cancer cells treated with inhibitors. One clear phenotypic difference is in the ratio of rods to rings in ESCs (rod∶ring = 1∶9) compared to all other cell types with induced RR and including the RR detected in INOVA HEp-2 slides (rod∶ring = 9∶1). It is unclear whether the spontaneous formation of RR in ESCs is related to the functional activity of IMPDH2 or CTPS1 but one can speculate that, since ESCs are metabolically highly active, the spontaneous formation of RR is likely to suggest that RR-associated IMPDH2 in ESCs are functionally active. The ability to self-renew and pluripotency are two unique properties of stem cells, and these properties may have an effect on IMPDH2 (and CTPS1) activity. Once stem cells differentiate, the cellular requirement of IMPDH2 (and/or CTPS1) may change and the RR is no longer needed. One biological function of RR, as demonstrated here, is related to CTP/GTP synthetic pathways.
Similar to ESCs, the INOVA HEp-2 cells were actively dividing cells (before fixation), based on the relatively high number of detectable mitotic cells. Thus, RR in INOVA HEp-2 cells are also likely to be functional in terms of their component CTPS1 and IMPDH2. One can speculate that the requirement of CTP and GTP in rapidly dividing cells favors the aggregation of these nucleotides synthetic enzymes to possibly enhance their functional activities. This is consistent with our experience that, while freshly thawed cells can demonstrate up to 25% cells with RR in first or second cell passages, cell confluence is often associated with the complete loss of RR expression – IMPDH2/CTPS1 no longer assemble in the cytoplasm. This argues for the formation of RR to be associated with a high requirement of intracellular CTP/GTP and the associated biosynthetic enzymes are assembled into RR, which can enhance their synthetic activity.
In contrast, inducible RR in cancer cells, as well as primary cells, are composed of inactivated enzymes based on the presence of an inhibitor added to induce these structures. Higher expression of IMPDH2 inhibited formation of RR by comparable levels of inhibitors. Lower expression of IMPDH2, as in the partial knockdown experiment, promoted formation of RR with a lower concentration of inhibitor. The apparent difference between ESCs with likely active RR-associated IMPDH2/CTPS1 versus the inhibitor-induced RR with inactive enzymes may be explained as follows. When HEp-2 cells are exposed to an inhibitor, such as Ribavirin, the lower production of GTP may trigger the response for the biosynthetic enzyme to assemble into oligomer/polymer configuration to favor an increase in enzyme activity, consistent with the speculation above that ESCs have a strong GTP/CTP requirement to maintain active cell proliferation. Once RR is formed, Ribavirin – an irreversible inhibitor, "locks" the configuration of RR, making the enzymes inactive.
In summary, RR formation is regulated by the inhibition of CTPS1 and IMPDH2, important in purine and pyrimidine biosynthesis and critical for RNA and DNA synthesis. The importance of these enzymes in RNA and DNA synthesis suggests that RR may play a role in cell division. In addition, both CTPS1 and IMPDH2 aggregate together to form the RR structure and inhibition of one will cause the aggregation of the other component with RR. Studies have demonstrated that CTPS1 and IMPDH2 are aberrantly expressed in different cancers [4], [29]. However, as CTPS1 and IMPDH2 are differentially expressed, the formation of RR in cells may help regulate their overall activities. Further characterization and understanding of RR may lead to a new biomarker for diseases or novel markers for stem cells.
Materials and Methods
Cell culture
HeLa (human cervical cancer), HEp-2 (human epidermoid larynx carcinoma), and HCT116 (human colon cancer) were cultured in DMEM containing 10% Fetal Bovine Serum (FBS). Human oral cancer cells CAL 27 were cultured in DMEM containing 10% FBS and 1.5 g/L sodium bicarbonate. THP-1, human monocytes, and K562, human erythroleukemia cells, were cultured in RPMI 1640 with 25 mM HEPES, and 10% FBS. All cells were cultured in a 37°C incubator with 5% CO2. Adherent cell lines were maintained at 50% confluence. Suspension cells were maintained at an optimum concentration of 106 cells/mL. All media contained 100 I.U./ml penicillin and 100 µg/ml streptomycin.
Embryonic stem cell culture and differentiation
The murine R1 ESCs were obtained from Dr. Andre Nagy (University of Toronto) and maintained according to Hamazaki et al. [30]. ESCs were differentiated with 1 µM retinoic acid for 4 consecutive days as described [31].
Mouse cardiomyocyte collection and preparation
The mouse cardiomyocytes were harvested and prepared according to Chan et al. [32].
Human autoantibodies
Autoantibodies with reactivity to RR were selected by authors GC and CAvM during routine ANA screening assay. All three human anti-RR sera used in this study were from patients with chronic HCV infection and were de-identified samples. These sera were selected largely due to their relative specificity in RR staining and in available quantity to allow further analyses. Serum It2006 provided by GC, and serum 604 and 609 provided by CAvM, are considered prototype sera for anti-RR. The clinical characterization of these patients and others with anti-RR will be summarized in a subsequent report [33].
Immunoprecipitation of [35S]-methionine-labeled K562 cells for the analysis of the proteins recognized by human autoimmune sera was performed as described [34].
Indirect immunofluorescence
HEp-2 cell slides (INOVA Diagnostics, San Diego, CA) or various adherent cell types cultured in 8-well chamber slides (BD Falcon) were used for indirect immunofluorescence as described [35], [36]. The 8-well chamber slides were prepared using ∼60,000 cells per well in 500 µL of media. Non-adherent cells were prepared using the cytospin method to transfer cells onto glass slides as described [37]. Fixation methods included acetone at −20°C for 5 min, acetone/methanol (3∶1) at −20°C for 10 min, and 3% paraformaldehyde in PBS at room temperature for 10 min followed by 0.1% triton-X/PBS for another 10 min. For co-staining studies the following primary antibodies were used: rabbit anti-giantin [38], anti-pericentrin (a gift from Dr. Marvin Fritzler, University of Calgary), anti-Drosophila CTPS1 and pre-immune serum (a gift from Dr. Jim Wilhelm, University of California, San Diego), anti-IMPDH2 (Abcam, Cambridge, MA: ab75790; Proteintech, Chicago, IL: 12948-1-AP), mouse anti-actin, anti-actinin (Sigma-Aldrich, St. Louis, MO: A7811), anti-tubulin, and anti-vimentin, and human anti-RR serum It2006, 604, and 609. To validate that the rabbit anti-Drosophila CTPS1 serum recognized human CTPS1, a full-length human CTPS1 cDNA (IMAGE id 3355881, OpenBiosystems, Thermo Scientific) was used as a template in an in vitro transcription and translation reaction (TNT, Promega) to generate [35S]-methionine labeled translation products. The latter was shown to be immunoprecipitated by the rabbit anti-Drosophila CTPS1 serum and not by the pre-immune serum from the same rabbit.
Induction of RR formation using CTPS or IMPDH inhibitors
Acivicin (Enzo Life Sciences, Ann Arbor, MI; BML-El113-0010) and Ribavirin (Sigma-Aldrich; R9644) were solubilized in water to 100 mM and 50 mM stock concentration, respectively. 6-diazo-5-oxo-L-norleucine (DON, Sigma-Aldrich; D2141) was solubilized in DMEM to a stock concentration of 100 mM. Cells were seeded as monolayers and allowed to attach for 24 h. Subsequently, CTPS1 or IMPDH2 inhibitors were added to various final concentrations from 0.2 µM to 2 mM and were kept in a cell incubator for times ranging from 15 min to 48 h.
siRNA knockdown
All siRNAs used in this study were obtained from Dharmacon RNA Technologies (Lafayette, CO). The siRNAs were dissolved in molecular biology grade water and the resulting 20 µM stock was stored in aliquots at −80°C before use. The two predesigned siRNAs were: ON-TARGET plus SMART pool siRNA Human CTPS1 (target sequences: UAACAUUGAUGCAGGAACA, AGACUAAACCUACCCAGAA, GGUGUUAUAUCAGGAAUUG, and GUAUGCAGGUGCUCAAAUC), siGENOME SMARTpool siRNA IMPDH2 (target sequences: GGACAGACCUGAAGAAGAA, GCACGGCGCUUUGGUGUUC, GGAAAGUUGCCCAUUGUAA, and CUAAAGAAAUAUCGCGGUA). Transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) per the manufacturer's instruction. The siRNA was used at a final concentration of 100 nM. Knockdown efficiency was determined by real-time PCR analysis using CTPS primers (Applied Biosystems, Hs01041851_m1) or IMPDH2 primers (Applied Biosystems, Hs00168418_m1) plus Western blot for protein expression.
Transfection of GFP plasmid
HeLa cells were seeded in 8 well chambers at 50–70% confluency. Cells were transfected with 0.35 µg GFP-IMPDH2 (RG202977, OriGene, Rockville, MD) or a GFP vector alone using 0.53 µg of Lipofectamine 2000. The transfected cells were examined after 24 h directly as live cells or were processed for indirect immunofluorescence.
Western blotting
Protein levels were analyzed as described [39] using rabbit anti- CTPS (Abcam, 1∶1000) or rabbit anti-IMPDH (Santa Cruz Biotechnology, Paso Robles, CA, 1∶1000). For IP-Western, IgG was crosslinked to protein A Sepharose beads as described [40] and human It2006 was used for immunoprecipitation and mouse monoclonal and rabbit anti-IMPDH from Santa Cruz Biotechnology were used for detection. Mouse anti-actin antibody 31G9 was used at 1∶5,000 or ∼1 µg/ml as a normalizer to control for loading.
Statistical analysis
Cell counting was performed using Mayachitra Imago (Mayachitra, Santa Barbara, CA) to detect nuclei counterstained with DAPI. Unpaired, two-tailed Student's t-test and Fisher's exact test were used to compare independent groups. When appropriate, Bonferroni's adjustment for multiple comparisons was applied. For all statistical analysis, Prism for Windows, version 5.0 (GraphPad Software, San Diego, CA) was used.
Erwin Lam, Natasha Deming, and Pabina Dhawan are acknowledged for the early work helping to develop this study. Dr. Joseph G. Gall, Department of Embryology, Carnegie Institution for Science, Baltimore, MD, is gratefully acknowledged for the very early consultation on the similarity of RR to structures he observed in GFP-CTPS Drosophila ovaries.
Author Contributions
Conceived and designed the experiments: WCC MS EKLC. Performed the experiments: WCC MS TH JYFC BY ST EKLC. Analyzed the data: WCC MS EKLC. Contributed reagents/materials/analysis tools: HK NT CAvM GC. Wrote the paper: WCC MS EKLC.
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- 17. Liu JL (2010) Intracellular compartmentation of CTP synthase in Drosophila. J Genet Genomics 37: 281–296.
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Blüthner M, Appelhans H, Moosbrugger I, Wiemann C, Lenz D, et al. (2009) Identification of inosine-1,2-monophosphate-dehydrogenase-2 as a novel autoantigen. In: Conrad K, Chan EKL, Fritzler MJ, Humbel RL, von Landenberg P, et al., editors. pp. 125–126. Proceedings 9th Dresden Symposium on Autoantibodies: Pabst Science Publishers, Lengerich, Germany.
- 19. Wu F, Lukinius A, Bergstrom M, Eriksson B, Watanabe Y, et al. (1999) A mechanism behind the antitumour effect of 6-diazo-5-oxo-L-norleucine (DON): disruption of mitochondria. Eur J Cancer 35: 1155–1161.
- 20. Shan G, Li Y, Zhang J, Li W, Szulwach KE, et al. (2008) A small molecule enhances RNA interference and promotes microRNA processing. Nat Biotechnol 26: 933–940.
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- 23. Ingerson-Mahar M, Briegel A, Werner JN, Jensen GJ, Gitai Z (2010) The metabolic enzyme CTP synthase forms cytoskeletal filaments. Nat Cell Biol 12: 739–746.
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- 32. Chan JY, Takeda M, Briggs LE, Graham ML, Lu JT, et al. (2008) Identification of cardiac-specific myosin light chain kinase. Circ Res 102: 571–580.
- 33. Covini G, Carcamo WC, Bredi E, von Muhlen CA, Colombo M, et al. (2012) Cytoplasmic rods and rings autoantibodies developed during pegylated interferon and ribavirin therapy in patients with chronic hepatitis C. Antiviral Therapy. in press.
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Nicene and Ante-Nicene Fathers, Ser. II, Vol. IV:Early Church Fathers Index Previous Next
Apology to the Emperor. (Apologia Ad Constantium.): How Athanasius acted when this took place.
26. How Athanasius acted when this took place.
Now why did they form this plot against me, and treacherously lay an ambush to take me, when it was in their power to enforce the order by a written declaration? The command of an Emperor is wont to give great boldness to those entrusted with it; but their desire to act secretly made the suspicion stronger that they had received no command. And did I require anything so very absurd? Let your Majestys candour decide. Will not every one say, that such a demand was reasonable for a Bishop to make? You know, for you have read the Scriptures, how great an offence it is for a Bishop to desert his Church, and to neglect the flocks of God. For the absence of the Shepherd gives the wolves an opportunity to attack the sheep. And this was what the Arians and all the other heretics desired, that during my absence they might find an opportunity to entrap the people into impiety. If then I had fled, what defence could I have made before the true Bishops? or rather before Him Who has committed to me His flock? He it is Who judges the whole earth, the true King of all, our Lord Jesus Christ, the Son of God. Would not every one have rightly charged me with neglect of my people? Would not your Piety have blamed me, and have justly asked, After you had returned under the authority of our letters, why did you withdraw without such authority, and desert your people? Would not the people themselves at the day of judgment have reasonably imputed to me this neglect of them, and have said, He that had the oversight of us fled, and we were neglected, there being no one to put us in mind of our duty? When they said this, what could I have answered? Such a complaint was made by Ezekiel against the Pastors of old 1358 ; and the blessed Apostle Paul, knowing this, has charged every one of us through his disciple, saying, Neglect not the gift that is in thee, which was given thee, with the laying on of the hands of the presbytery 1359 . Fearing this, I wished not to flee, but to receive your commands, if indeed such was the will of your Piety. But I never obtained what I so reasonably requested, and now I am falsely accused before you; for I resisted no commands of your Piety; nor will I now attempt to return to Alexandria, until your Grace shall desire it. This I say beforehand, lest the slanderers should again make this a pretence for accusing me.
Ez. xxxiv. 2, &c.248:1359
1 Tim. iv. 14.
Next: Athanasius leaves Alexandria to go to Constantius, but is stopped by the news of the banishment of the Bishops.
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You are here
Successfull Information Evening 2017
This year's Information Evening for the pre-IP and the IB Diploma Programme (IB DP) was kicked off with a great performance by our talented Showband.
Headmaster Jens Boe Nielsen then gave an introductory welcome, and IB Coordinator Jutta Rüdiger followed up with her warm welcome to all the visitors – potential future students and their parents.
After introducing the international programmes in general, the Coordinator continued with specific information about the pre-IB assisted by Charlie and Chrysanthi from our 1j class whose narratives helped illustrate the student perspective of the start and daily life at Nørre Gymnasium.
The IB DP, our two-year education, was up next. Here, the Coordinator's information was complemented by Emil and Isabel, both IB DP students in the second year soon to endure the final exams.
Anne Wilson, our IB Student Counsellor, told the visitors about our student counselling specifically oriented at IB students and international studies as well as the many aspects of students' lives where they can get help from the counsellor.
A great line-up of students representing some - but far from all - of the extracurricular activity groups at Nørre G.
In the Foyer, we had booths manned by engaged teachers and students presenting all the subjects and other information vital for making choices regarding an education in the International Programmes at Nørre Gymnasium.
Other students acted as guides to lead the visitors giving them a feel of the school itself and the learning environment.
We thank you all for a great evening and hope to see many of the visitors as students in the pre-IB or the IB DP at Nørre G in August.


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Well after 26 months of pregnancy, I am finally starting to eat like a pregnant woman (Some may say I have always eaten this way, but it has been worse in the last week or so). I just cannot get full, plus Easter candy is out and, oh my, how I love Easter candy. I am craving sweets and ice cream. I really need to get this under control or we may have to get an extra seat for my butt on the way to China. During the wait I have maintained a pretty consistent (although over the limit) weight. It has gone down a couple of times but usually ends up bouncing back to the original number. I the pattern I have been following the last couple of days continues, I am going to go very the usual and that is just too much. I need to lay off the jelly beans and find some fruit and vegetables. I really want to be able to climb THAT Wall before we get Annika (Luke) and now that I won't make it too far in my current condition.
(A funny note: when I proof-read my post I had spelled weight "wait" in the title.)


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In honor of Mickey Mouse's 85th birthday, we're going to be saluting Mickey all this week.
First up: From Season 3 of Disney's House of Mouse comes a 2001 short that made its network television premiere on the show: "Mickey's Airplane Kit". Mickey's totally forgotten his date with Minnie, and has to improvise his way out of possible trouble.
Mickey & Minnie have been one of toondom's most enduring couples, much like Popeye and Olive Oyl. While Olive & Popeye made it to the altar in a 1-shot comic book special some 15 or so years ago, Mickey & Minnie haven't yet said "I do!", and maybe that's part of the charm.
Now, here's "Mickey's Airplane Kit":
Earlier this year, Disney began producing new shorts for television, one of which was reviewed in this space previously. Figure on more during the week.
No rating for "Mickey's Airplane Trip", as I didn't see it when it first came out.


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Bruce Springsteen Crashes David Geffen's Documentary Premiere
The Boss was a walk-in at the mogul's star-studded Nov. 13 event, featuring Cher, Tom Hanks, Steven Spielberg and Warren Beatty.
This story first appeared in the Nov. 30 issue of The Hollywood Reporter magazine.
It's clear that David Geffen's the boss from the PBS American Masters documentary Inventing David Geffen: The man invented modern moguldom.
PHOTOS: 20 Biggest Political Players in Hollywood
But at the L.A. premiere Nov. 13 at the Writers Guild Theater, hosted by Cher, Tom Hanks, Rita Wilson, Steven Spielberg, Kate Capshaw, Warren Beatty and Annette Bening, there was another boss present: Bruce Springsteen.
Organizers tell THR he was "a walk-in." When a well-wisher congratulated him on helping to re-elect President Obama, Springsteen actually blushed.
"I only had a little, little bit to do with it," he tells THR.
STORY: 'Inventing David Geffen' Draws Rare A-Plus Crowd to L.A. Premiere
Meanwhile, at a Spago dinner afterward, some feelings were hurt when organizers apparently kept some of Geffen's pals off the list.
Geffen himself arrived with date Oprah Winfrey and claimed to have nothing to do with the dinner guest list.


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13 Suzuki RM-Z250 Financing Sales (847) 526-0500 315 N. Rand Road Wauconda Illinois 60084 Video 1 of 26 View Large Images Options and Standard Features Basic Information Year: 2013 Make: Suzuki Model: RM-Z250 VIN: 4574460 Condition: Used Type: Standard Mileage: 9999 Title: Clear Color: Champion Yellow Engine Engine Size (cc): 0 Specifications Length (inches): 85 Contact Monster Powersports 315 N. Rand Road Wauconda Illinois 60084 View Map Ask for:Sales Main:(847) 526-0500 Contact: Request Quote Hours Mon-Fri:10:00 AM-07:00 PM CST Saturday:10:00 AM-05:00 PM CST Sunday:Closed Directions to Dealer Request Quote Vehicle Information Year: 2013 Make: Suzuki Model: RM-Z250 Condition: Used Financing Terms Placing a Bid: Your bid constitutes a legally binding contract to purchase this vehicle. Please do not bid if you're not seriously interested or financially able to purchase this vehicle. Please read eBay's User AgreementMonster Powersports reserves the right to, Obtain and verify the registered information of all users who bid on this auction. Cancel any and all bids at our discretion, or end the auction early if necessary.Bidders Age: You must be 18 years of age or older to Bid. Special eBay Bid Retraction Rules: Please read eBay's "Retracting a Bid" If you place a bid before the last 12-hour period of the auction: You may retract that bid before that last 12-hour period but only for exceptional circumstances. You will not be allowed to retract that bid during the last 12-hour period of the auction. If you place a bid during the last 12-hour period of the auction: You will be allowed to retract the bid for exceptional circumstances but only if you do so within one hour after placing the bid.Funds & Financing: For help in arranging for a Pre-Approved loan or for any questions please e-mail or phone at(847) 526-0500 prior to bidding. Buyers Inspection: Monster Powersports has done our best to disclose all information known about this vehicle for auction. Monster Powersports welcomes a buyers inspection. If you plan to have a buyers inspection, please make sure you inspect the vehicle prior to the auction ending. Inspection fees if any are Buyers responsibility. Representations and Warranties made by seller: This vehicle is being sold "as is". Manufacturer's warranties may still apply. Extended warranty may be available, e-mail or phone at (847) 526-0500 for details. No representations or warranties are made by seller, nor are any representations or warranties relied upon by bidders in making bids. Taxes and Registration fees: Out of state buyers are responsible for all state, county, city taxes and fees, as well as title service fees in the state that the vehicle will be registered. All taxes and fees must be paid in full in order for vehicle to be titled and registered. Title Information: Vehicles titles may be held by banks or lenders as collateral for loans. In many cases there is a delay in receiving the original instruments up to 21 days from the time we pay a vehicle off. While we usually have all titles in our possession at closing, there are occasions where we may be waiting for them to arrive. If payment is made by cashier's or personal checks we will hold all titles for 10 days or until funds have cleared. Shipping & Delivery: All shipping charges are buyer's responsibility. Monster Powersports will help with shipping arrangements but will not be responsible in any way for claims arising from shipping damage!Licensed Carriers are generally insured for $3,000,000.00. We assume no responsibility for damages incurred after the vehicle leaves our showroom. All shipping arrangements are provided by Monster Powersports as a courtesy. We are not affiliated with any carrier. Any claims or other communication regarding shipment of vehicles will be between you and the shipper, not with Monster Powersports. The amount of time it takes for delivery is dependent on the carrier, but is generally 7-14 days from the date the vehicle is picked up from our facility until it is delivered to your destination. Verify with the shipper for an Estimate Time of Arrival to be sure. Finalizing your Purchase: Monster Powersports will contact the successful high bidder by e-mail after the auction closes. Successful high bidder MUST communicate with at Monster Powersports by e-mail or phone(847) 526-0500 within 24 hours of the auction ending to make arrangements to complete their transaction. If we cannot confirm your intention to buy or the sale is not completed within 5 days, we reserve the right to relist this vehicle or sell to any other qualified buyer. In order to secure bid on vehicle, Successful bidder (BUYER) must within 24 hours of bid closing send to Seller a Deposit in the amount of 10% by major credit card, cash in person or bank certified funds. Within 72 hours of bid closing, Buyer must send balance of funds by bank wire transfer, cash in person, bank certified funds to Seller. At time of sending initial deposit, Buyer MUST fax copy of their State issued valid Driver License. Furthermore, before said vehicle is released for shipment to Buyer, all other Sale related and title related paperwork must be signed and returned complete to Seller. Copyright © 2016 Auction123 - All rights reserved. - Disclaimer +- Auction123 (a service and listing/software company) and the Seller has done his/her best to disclose the equipment/condition of this vehicle/purchase. However, Auction123 disclaims any warranty as to the accuracy or to the working condition of the vehicle/equipment listed. The purchaser or prospective purchaser should verify with the Seller the accuracy of all the information listed within this ad. Selling a Vehicle? Create Professional Listings Fast and Easy. Click Here!


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Environmental Policy
The Scottish Crannog Centre is dedicated to operating and educating in an environmentally responsible manner.
We recycle building and other materials, harvest timber and plants in accordance with sustainable woodland management agreements and we sell eco-friendly products in our giftshop.
We encourage all staff to buy Fair Trade and organic products wherever possible. In addition, we aim to be informed about local amenities and sites of cultural and environmental interest so that we may give useful advice or recommendations to both residents and visitors to the area. As a result of these and other policies, we gained the prestigious GOLD Award from the Green Tourism Business Scheme in Scotland. The scheme promotes best practice and encourages businesses to reduce their impact on the environment. To view thier website, please click here.
Visitor Charter
We hope to encourage all visitors to share our passion for the environment and do what they can to reduce impacts.
Our Environmental Policy
Statement: Our 'official' environmental statement covers relevant areas of administration, energy use, giftshop ethics and green suppliers, the crannog itself, our activities area, refreshment kiosk, transport issues, waste management, and communication practices. Our Policy can be downloaded below.
We are people people: we work closely with local businesses and individuals especially in joint special events; we promote local producers and craftspeople in our giftshop and cafe, and we like to think we live as responsibly as our ancestors did. We are also members of a number of networks and NGOs that promote both our natural and cultural environment, responsible foraging and woodland management. Why not come and meet us to find out more?
We encourage visitors to take public transport. Details of how to get to the Centre using buses, trains or on a push-bike can be found on our contact page
Green Projects
The ancient crannog dwellers were great users of willow and hazel. For thousands of years these woods used to be coppiced and well-managed in Perthshire, but the practice died out some 30 years ago. As we are also great users of these woods, we already plant willow but we initiated the Perthshire Coppice Project to regenerate or to establish new hazel coppice woodlands in conjunction with local estate owners and woodland organisations. The benefits include improved habitats for wildlife, sustainable products, rural skills training, and job opportunities. If you are interested in supporting this work, please contact us.
We run many themed events which focus on the crannog-dwellers and their interaction with their environment. We showcase a range of plants which were found in our underwater excavations and provide information on their culinary, medicinal and dyeing properties, while food events focus on wild food.
We are proud to be members of Scottish Wild Harvest Association and supporters of Scottish Working Woods, Reforesting Scotland, The Star Tree Project, Tayside Biodiversity and Perth and Kinross Countryside Trust.
Scottish Crannog Centre Environmental Policy


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Cub Scouting is the foundation of our organization. And while it is designed for boys in the first through fifth grades, this program really involves the whole family as parents pitch in to plan and deliver activities. Cub Scouting is affirmative and fun focused. There are currently 1.3 million Cub Scouts and 395,000 adult volunteers in the United States.
Cub Scouts is a year-round program uniquely designed to meet the needs of young boys ages 7-10 and their families. The program offers fun and challenging activities that promote character development and physical fitness. Boys can begin as Tigers as they finish Kindergarten, and then move into the Wolf and Bear Cub Scout programs (second and third grade, or age 8 or 9), and Webelos Scouts (fourth grade and fifth grade, or age 10). They then transition into the Boy Scout Program.
When you're a kid, fun comes first. Final exams, driver's ed and summer jobs will arrive soon enough. Cub Scouts lets kids have fun while teaching them skills that will help them later on. From the thrill of shooting an arrow to learning how to transform a block of wood into a race car, Cub Scouts is one thrill after another. With a program expertly tailored to match each age in a young boy's life, Cub Scouts learn that even when fun isn't easy, it's always an adventure. In Scouting, everything has a purpose. And it shows kids that doing their best is the most fun of all.
Cub Scouts are broken into a small group called a pack. Each pack has dedicated leaders and volunteers who are tasked with teaching Cub Scouts both fun and valuable lessons that will help them progress in the program and in life. But what makes Cub Scouts truly unique is the opportunity it gives the entire family to be involved in each child's development. From volunteering with the pack to leading activities at home, parents can take full advantage of the extra time they'll have participating in their Cub Scout's life! The pack you join is up to you. You can look for a pack chartered by an organization you support, or simply choose the one closest to your neighborhood.
To find a Cub Scout Pack near you visit:
This Web site will help you learn all about Cub Scouting and how you can join in the fun!
Scouting Safely
Training Times
BSA Adult Training
Family Scouting
GNFC eNews Signup
Order of the Arrow Yearly Dues
GNFC Honor Roll
Website Questions/Comments email us at firstname.lastname@example.com
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Locarno Announces Plans to Upgrade Filmmakers of the Present Sidebar
Bigger prizes and competitive format are the main changes to the event for emerging directors.
ROME – The Locarno Film Festival announced Friday that it would revamp and upgrade its Filmmakers of the Present sidebar to increase its visibility, making it the festival's second competitive section.
The change will also increase prizes to winners, including a new prize: the Best Emerging Director prize, worth 40,000 Swiss francs ($45,000), an increase of a third compared to last year's top prize in the sidebar. The sidebar's second prize, for the top emerging director, will be worth half as much as the main prize.
The Filmmakers of the Present sidebar will be made up of about 15 feature films and documentaries, all world or international premieres and all from young directors with their first or second production. It will join Locarno's prestigious main competition section as the festival's only competitive sections.
The 65th edition of picturesque lakeside Swiss festival will take place this year Aug. 1-11.


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Andrew Driver is the Club's professional, having taken over from John Hammond at the start of October 2015. Prior to this, Andrew served as Assistant Professional to John for 11 years and offers a friendly welcome to members and visitors to the Club. He has a wide knowledge of the game of golf and the latest equipment, whilst he has developed a reputation as a very good coach to golfers of all ages and abilities.
Information about the Professional Golf Shop
The Professional Shop has recently undergone refurbishment and now has a new improved layout offering a first class retail experience and is stocked with the leading equipment and clothing brands.
Andrew is a first class teacher and he and his team are available for lessons for both members and non-members. Everyone is welcome whatever standard. Group coaching is also available.
He can also help you to organise your golf day from suggesting the format and running the competition, through to providing the prizes for the winners.
The hire of trolleys, two seater buggies and golf clubs may also be arranged with the professional.
Here are some Special Offers on the golf course.
Golf Lessons
Andrew £25 per half hour
By booking a course of 6 lessons with Andrew, you get 1 free, so 6 for the price of 5.
Andrew can use video analysis and the latest teaching equipment called the SkyPro.
The SkyPro is quite simply the ultimate training aid, it has the power to dramatically improve your game and make the most of your lessons.
Skypro Features
3D playback of your swing.
View your swing from all angles in real time or in slow motion.
Store and view thousands of swings.
Synchronise and compare two swings.
Built in alerts to help identify swing faults at certain points in the swing.
The SkyPro allows the player to work on swing tempo, shaft lean and angles at address and impact, backswing length, clubhead speed drills, swing path, angle of attack, face rotation and much more.
Contact Details
Pro Shop 01943 607463 or email email@example.com


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I truly look forward to these times. There is no technology, no phones, cells, tv nor gaming devices. We stay in a rustic log cabin that is outfitted with reclaimed camper appliances (stove and mini fridge). I boil water to do dishes and by time I am done cleaning up one meal it is nearly time to begin the next lol.
I will take the kids fishing, we will go on nature hikes and generally just hang out. The best part....the view! I LOVE to pull a chair onto the tiny deck and sit knitting while watching the kids at play.
Once I return, there will be a "Scrappy" stocking on Etsy for Sept 5th. Look for gems like this beautiful rainbow AIO to be listed.