News Article | April 29, 2017
The 2017 Silicon Labs Sunshine Run is quickly approaching and race organizers are proud to now carry the mantle of Austin’s Most Charitable 5K/10K Event. Runners and walkers from across Central Texas will descend upon Vic Mathias Shores (formerly Auditorium Shores) for the 4th Annual Silicon Labs Sunshine Run, benefiting the Austin Sunshine Camps. The run features a 5K, 10K, Kids K, and ‘Fastest Dog in Austin 5K’. The top 3 male and female finishers in each age group for the timed 5K and 10K races will receive medals. All participants will receive technical shirts and race bibs during packet pickup on May 5th and 6th. All 5K and 10K runners will start at Vic Mathias Shores near the corner of Riverside Dr and South 1st Street. Runners will head north across the 1st Street bridge, and hang a left on Cesar Chavez Street, passing the corporate headquarters of title sponsor Silicon Labs. Runners will race west on Cesar Chavez Street and then veer off to the left at Austin High School on Stephen F Austin Drive. 5K participants will loop under Mopac and then turn back east on Cesar Chavez to re-trace their route back to a cheering crowd at the finish line at Vic Mathias Shores. Those participating in the USATF-certified 10K will continue passed Austin High School on Stephen F Austin Drive and Veterans Drive. They will continue west on Lake Austin Blvd before the halfway point turnaround at Exposition Blvd. The Kids K is a one kilometer course that loops along Riverside Drive just in front of the Long Center so that parents can supervise their children. All kids participating in the Kids K will receive a finisher’s ribbon. Austin-based Nulo Pet Food is proud to once again sponsor the ‘Fastest Dog in Austin 5K’. The top male and female finisher of the timed 5K with a registered dog will receive a year’s supply of Nulo Pet Food. In addition, for each dog that visits the Nulo tent on race day, Nulo will donate 5 meals to the Austin Animal Center and Nulo has a treat for each dog that stops by. With this flat and fast course being USATF certified, race organizers are expecting a large group of competitive runners. However, event organizers are quick to point out that this run is all about creating a welcoming environment for active families from across Austin and that the spirit of the day is celebrating the Austin Sunshine Camps. And that is precisely why organizers can now say they are Austin’s Most Charitable 5K/10K Event. “This run is so much fun for everyone that comes out, but we are most excited for the direct impact that this run makes on kids in our community,” notes Jace Campbell, marketing director for the run and owner of PrimeFirst Inspections. “Because of our generous sponsors underwriting our race day expenses and because the Young Men’s Business League provides a 100% volunteer work force, we can now say that more dollars per race entry goes back to our beneficiary than any other 5K / 10K event in Austin.” Last year’s event brought out over 2,500 participants and race organizers are anticipating an even larger turnout this year. “As we prepare for summer and the 1,200 kids we will host at camp, we are very excited for the community to come enjoy this beautiful downtown run while we raise money for the camps,” says Jorge Padilla, co-director of the Silicon Labs Sunshine Run and Board member of the Young Men’s Business League. “We welcome strollers, dogs on leashes, walkers, runners, joggers. Come one, come all. You won’t regret taking part in this event.” Online registration is currently open at AustinSunshineRun.com and online registration closes at 7pm on Thursday, May 4th The Austin Sunshine Camps have served over 48,000 disadvantaged youth since its founding in 1928. Participation in summer learning programs such as the Austin Sunshine Camps helps combat summer learning loss, which disproportionately impacts low-income youth. More information on the Austin Sunshine Camps is available at SunshineCamps.org. For more information, please visit https://primefirstinspections.com
Di Cristina G.,Stazione Zoologica Anton Dohrn |
Andrews P.,University of London |
Andrews P.,Association for Cephalopod Research |
Ponte G.,Association for Cephalopod Research |
And 4 more authors.
Invertebrate Neuroscience | Year: 2015
Here we discuss several impacts of some of the changes that have occurred since the implementation of Directive 2010/63/EU and of other regulations and directives on the scientific research involving cephalopods. Changes that correspond to a significant turning point of policies require responses from all those involved in research with cephalopods (including aspects of aquaculture research) as well as those responsible for their daily care and welfare. © Springer-Verlag Berlin Heidelberg 2015.
Lin Y.-H.,National Cheng Kung University |
Chou C.-K.,Animal Center |
Hung Y.-C.,Animal Center |
Yu I.-S.,National Taiwan University Hospital |
And 3 more authors.
Fertility and Sterility | Year: 2011
Oocytes fertilized with spermatozoa obtained from Septin 12+/- chimeric mice failed to develop beyond the morula stage after IVF and intracytoplasmic sperm injection because of significant DNA defects in the spermatozoa. Given that SEPT12 is expressed at the edge of the sperm nucleus in both humans and mice, we hypothesized the vital roles of Septin 12 in sperm head shaping, nuclear DNA condensation, and early embryonic development. © 2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
News Article | August 26, 2016
Even on sweltering summer days, the popular smartphone game has gotten throngs of players out of their homes to real-world locations designated as "PokeStops" and "Gyms." Theme parks, bars and even a county animal shelter are among those trying to capitalize on that surge in foot traffic. In New York, Doughnut Plant created an edible version of the Poke Ball—dubbing it the Pokeseed—after a Pokemon-obsessed employee realized that all four of the company's shops are either PokeStops or very close to one, owner Mark Isreal said. And one location is an in-game Gym, making it a gathering place to both consume and virtually burn off calories. The team at Doughnut Plant designed the fruity treat in less than a day, using cranberry-raspberry and white chocolate icings to recreate the red-and-white Poke Balls, the objects used in the game to capture monsters. The Pokeseed is stuffed with a peach-strawberry cream filling, an imagining of Pokemon's mythical pecha berry. Pictures went out on social media the next morning, "and before they were delivered, people were already coming to the stores," Isreal said. Doughnut Plant has already sold thousands of Pokeseeds, and customers frequently post pictures of them on Instagram. They're still selling strong, so Doughnut Plant has no plan to take them off the menu any time soon. Meanwhile, a trendy food court near New York's Penn Station put up a sign urging passersby to catch a Pokemon instead of a train, while the city's parks department created "PokeFit" classes for kids to play while exercising. Earlier, the Busch Gardens theme park in Florida hosted a Pokemon "lure-a-thon," with some PokeStops accessible only by season-pass members for one hour. The Pawtucket Red Sox baseball team in Rhode Island invited fans onto the field to chase the virtual monsters. Police in Manchester, New Hampshire, even tried to lure fugitives by claiming to have detected a rare Charizard in the booking area. A Facebook post invited those on a list of "lucky ones" to capture the monster—the list happens to be filled with the city's most wanted. Andy Wong of Kurt Salmon Digital, which helps retailers connect digitally with consumers, said the game has worked well for small businesses, though there hasn't been a good way for larger companies with hundreds of stores to automate the "lures" they buy to attract digital monsters—and with them, players and potential customers. And even for small businesses, he said, the ability to draw customers may have diminished as the game loses its novelty. But those that caught the bug early saw tangible benefits. The Phoenix Zoo was a hotbed of Pokemon activity right after the game's release last month, even when temperatures climbed as high as 112 degrees. It helped that a Pokemon Gym was housed in the zoo's conveniently air conditioned orangutan house. After noticing that some visitors were on the hunt for more than just traditional zoo creatures, the zoo opened an hour early at 6 a.m. for a week during what's usually a slow time of year. The zoo also converted its train into a "PokeShuttle" that pointed out PokeStops along with its animal exhibits. On the first day of the promotion, attendance more than doubled from a week earlier, and sign-ups for new memberships spiked, said zoo spokeswoman Kerri Baumann. "It has snowballed in the most exciting and fun way," she said. Given the popularity, zoo officials are considering having additional Pokemon-themed activities, she said. Other furry creatures have benefited, too. The Wake County Animal Center in Raleigh, North Carolina, said its Pokemon-themed social media posts prompted about 25 applications for volunteer dog walkers, about four times what it usually gets. "If people are getting out and walking, why not come out here and walk the dogs and catch some Pokemon?" said Jennifer Federico, Wake County's animal services director. "It's fun and it gets people out." The shelter also named dogs and cats after Pokemon characters in hopes of giving animals that may get overlooked a second chance at adoption, she said. Bars and restaurants are getting in on the action as well, both through numerous Pokemon-themed bar crawls around the country and by taking advantage of nearby stops and gyms on their own. Because street art accounts for a substantial number of PokeStops, especially in big cities, the Tyron Public House bar and restaurant in New York has seen a slight bump in business, thanks to a large mural outside. Some patrons have paid for lures to attract more Pokemon; others return the favor by buying them drinks. "It's kind of fun to see people playing and say, 'Here you go. Enjoy,'" Tyron manager Errol Flynn said. "For us, it's not so much about organized events as much as it is about keeping up with social and what's going on in the neighborhood." Explore further: How to play Pokemon Go
News Article | November 4, 2016
PLEASANTON, CA--(Marketwired - November 03, 2016) - Maddie's Fund®, a national family foundation based in Pleasanton, CA, awarded the first annual Avanzino Leadership Award to Dr. Ellen Jefferson of Austin Pets Alive! for her outstanding leadership and significant achievement in lifesaving. The award is named after Rich Avanzino, the father of the no-kill movement, and recognizes outstanding leadership in the animal welfare community. It will be presented along with a $25,000 grant to Austin Pets Alive! for demonstrating significant achievement in lifesaving, showing the courage to look beyond the status quo and making bold decisions to improve the lives of dogs and cats, and being a champion of the no-kill movement. "Dr. Ellen Jefferson, Executive Director, Austin Pets Alive!, embodies these qualities and more," said Amy Zeifang of the Maddie's Fund Executive Leadership Team. "She was the overwhelming choice as Maddie's Fund's first recipient of this award." In 2008, Dr. Jefferson stepped in as Executive Director of Austin Pets Alive! in an effort to expedite achieving no-kill in their community. She formed collaborative ties with Austin Animal Center and Austin Humane Society. Together, the organizations brought the entire city of Austin to a greater than 90% save rate, and became the largest No-Kill city in the U.S. In 2012, Dr. Jefferson helped San Antonio Pets Alive! to implement the no-kill programs that were proven successful in Austin and helped drive the live release rate from 30% to 80% in 12 months. Moving forward, the Avanzino Leadership Award will be awarded to one person each year through peer nominations. The nominations will be accepted in July/August with an announcement of winner in September 2017. Animal welfare practitioners are invited to nominate their choice for who best embodies the qualities of the Avanzino Leadership Award, and are encouraged to join the Maddie Network for updates. Maddie's Fund® is a family foundation created in 1994 by Workday co-founder Dave Duffield and his wife, Cheryl, who have endowed the Foundation with more than $300 million. Since then, the Foundation has awarded more than $187.8 million in grants toward increased community lifesaving, shelter medicine education, and pet adoptions across the U.S. The Duffields named Maddie's Fund after their Miniature Schnauzer Maddie, who always made them laugh and gave them great joy. Maddie was with Dave and Cheryl from 1987 - 1997 and continues to inspire them today. Maddie's Fund is the fulfillment of a promise to an inspirational dog, investing its resources to create a no-kill nation where every dog and cat is guaranteed a healthy home or habitat. #ThanksToMaddie.
News Article | December 21, 2016
The antibodies used for western blotting included anti-YAP/TAZ (1:1,000; 8418; Cell Signaling Technology, USA), anti-YAP (1:1,000; Cell Signaling Technology, USA), anti-pYAP (1:1,000; Ser 127, 4911S; Cell Signaling Technology, USA), anti-TAZ (1:1,000; ab84927; Abcam, UK), anti-JNK (1:1,000; 9252h; Cell Signaling Technology, USA), anti-pJNK (1:1,000; 9255; Cell Signaling Technology, USA), anti-CTGF (1:1,000; ab6992; Abcam, UK), anti-Gα (1:1,000; ab128900; Abcam, UK), anti-integrin β (1:1,000; 4702; Cell Signaling Technology, USA), anti-RhoA (1:1,000; ab54835; Abcam, UK) and anti-eNOS (1:1,000; BD Biosciences, USA). The antibodies used for immunostaining included anti-pYAP (1:100; Ser 127, 4911S; Cell Signaling Technology, USA), anti-YAP (1:100; Cell Signaling Technology, USA) and anti-pJNK (1:100; 9255; Cell Signaling Technology, USA). RNA was extracted by using TRIzol Reagent (Thermo) according to the manufacturer’s protocol. cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Thermo). Quantitative PCR was performed using SYBR Select (Thermo) following the manufacturer’s protocol. GAPDH was used as the internal control. Primers used for quantitative real-time PCR were included in Supplementary Table 1. Cells or tissues were homogenized in cold RIPA lysis buffer supplemented with cOmplete Protease Inhibitors cocktail and phosSTOP phosphatase inhibitor (Roche). The protein concentration was determined using Bradford Assay (Bio-Rad). Ten micrograms of protein were resolved by SDS–polyacrylamide gel electrophoresis and transferred to the PVDF membrane (Bio-Rad). Target protein was detected using specific primary antibody. Bound antibodies were detected by horseradish-peroxidase-conjugated secondary antibody and visualized by enhanced chemiluminescence (Cell Signaling Technology). Experiments were repeated three times and the target protein level was quantified by ImageJ and normalized to internal control (or pYAP was normalized to total YAP) (Extended Data Figs 6 and 7). Original western blot scans are included in Supplementary Fig. 1. HUVECs and human aortic ECs were purchased from Lonza (EGM, Clonetics, Lonza, Walkersville, Maryland, USA). Lonza guarantees that the cells express CD31/105, von Williebrand Factor VIII, and are positive for acetyated low-density lipoprotein uptake. We did not test for mycoplasma contamination during the experiments. HUVECs were maintained in EGM supplemented with EGS and FBS at 37 °C in an incubator with 95% humidified air and 5% CO and passaged every 3 days. Cells within seven passages were used for the in vitro study. GST-RBD recombinant protein was purified from BL21 (DE3) Escherichia coli and affinity conjugated to glutathione sepharose beads (Pharmacia). For GST affinity pull-down, 107 cells were lysed in 1 ml Weak Lysis Buffer (Beyotime) supplemented with protease inhibitors (Roche). Cell lysates were centrifuged at 15,000 g at 4 °C for 20 min to remove cell debris. Cell lysates were incubated in sepharose beads conjugated with 1 μg GST–RBD and incubated at 4 °C for 2 h with constant agitation, and precipitated by centrifugation at 1,000 r.p.m. for 10 min. After three washes, beads were collected by centrifugation and boiled in 2× SDS loading buffer for 5 min. The active RhoA was determined by western blotting. Animals were supplied by the University Laboratory Animal Services Centre and their use approved by the Ethical Committee of Animal Research (CUHK). The animals used in the present study included Sprague-Dawley rats, apolipoprotein E deficient (ApoE−/−) mice and EC-specific YAP overexpression transgenic mice. Male mice or rats were used in all in vivo studies. The animals were kept at a constant temperature (21 ± 1 °C) under 12/12-h light/dark cycle and had free access to water and standard chow unless specified. CAG loxp-stop-loxp-Yap mice were generated in a C57BL/6 background in Model Animal Research Center (Nanjing, China). Yap-COE mice were crossed with ApoE−/− mice and then Tie-2-Cre+/− mice. The 6-week-old ApoE−/−;Yap-COE;Tie-2-Cre+/− and ApoE−/−;Yap-COE;Tie-2-Cre+/− mice were bred and housed in temperature-controlled cages under a 12/12-h light/dark cycle with free access to water in Tianjin Medical University Animal Center. Study protocols and the use of animals were approved by the Institutional Animal Care and Use Committee of Tianjin Medical University (Tianjin, China). The mice were fed a Western diet (Research Diets, D12109) containing 40 kcal% fat, 1.25% cholesterol and 0.5% cholic acid for 4 weeks before being killed. Aortas were isolated to assess lesion formation and distribution by Oil Red O staining. Aortic roots were stained for pJNK, α-SMA and macrophages. Mouse aortas were fixed with 4% paraformaldehyde for 15 min. After permeabilization/blocking in 0.05% Triton X-100 (in PBS) and 1% BSA and for 0.5 h at room temperature, aortas were incubated at 4 °C overnight in incubation buffer containing 1% BSA and the primary antibody including YAP1 (Abcam, ab52771), CD31 (Abcam, ab24590). After being washed in PBS three times, aortas were incubated with Alexa-Fluor 488-, Alexa-Fluor 594-conjugated secondary antibodies (ZSGB-BIO, Beijing) for 1 h at room temperature. The fluorescent signal was detected by a Leica confocal laser scanning microscopy. Stenosis of the abdominal aorta of rats was induced using a U-shaped titanium clip, as described29, 30. Briefly, after anaesthetization with isoflurane, the rat was laid supine and a lower midline abdomen incision was made; the part of the intestine was gently lifted out of the abdominal cavity and kept moist with saline throughout the surgical procedure. The aorta, left and right common iliac artery were exposed and the accompanying vein was carefully separated. The clip was held with a pair of forceps and placed around the isolated segment (1 cm from the arterial bifurcation) to partly constrict the abdominal aorta. The extent of clipping was controlled by placing a stopper of given size between the two arms of the forceps. Two weeks later, the rat was euthanized by intoxication with 100% carbon dioxide, and the aorta was perfusion-fixed with 4% (w/v) paraformaldehyde at 120 mm Hg. The fixed aorta was embedded in paraffin blocks for immunohistochemical staining. Partial ligation of carotid artery was generated as described before31. Briefly, ApoE−/− mice were anaesthetized by intraperitoneal injection of xylazine (10 mg/kg) and ketamine (80 mg/kg) mixture. A ventral midline incision (4–5 mm) was made in the neck. Left carotid artery was exposed by ventral midline incision (4–5 mm) in the neck. Left external carotid, internal carotid and occipital arteries were ligated, while the superior thyroid artery was left intact. Mice were monitored until recovery in a chamber on a heating pad after surgery and fed the Western diet immediately after surgery until killed. Immunohistochemical staining was performed on serial sections (5 μm thick) of paraffin-embedded rat abdominal aortas and ApoE−/− mouse aortas using pYAP (Cell Signaling), EC- and SMC-specific markers (that is, vWF and α-SMA, respectively) (Merck Millipore). Briefly, the sections were de-waxed in xylene, rehydrated in descending grades of alcohol and permeabilized by incubating for 10 min in sodium citrate for 10 min at 95 °C. Sections were cooled down to room temperature and blocked with blocking reagent (Merck Millipore) for 30 min. One section was incubated with antibody against pYAP (1:100) overnight at 4 °C, followed by Alexa-Fluor 594-conjugated goat-anti-rabbit IgG (1:1,000; Invitrogen) secondary antibody in blocking reagent for 1 h at room temperature. The secondary section was incubated with antibodies against vWF and α-SMA (1:100 each) overnight at 4 °C, followed by Alexa-Fluor 594-conjugated goat-anti-rabbit IgG and Alexa-Fluor 488-conjugated goat-anti-mouse IgG (1:1,000; Invitrogen) secondary antibodies in blocking reagent for 1 h at room temperature. Nuclei were co-stained by DAPI (Invitrogen) in PBS for 5 min. The sections were spin-dried and mounted with ProLong Gold (Invitrogen) on glass coverslips. Images were acquired and analysed using a Zeiss fluorescence microscope with Axiovision image analysis software. ApoE−/− mice (male, 12 weeks old) were fed a Western diet, and MnCl was administered through voluntary water consumption. Water consumption rate was predetermined by monitoring the volume of water remained. MnCl was supplemented to drinking water to achieve 5 mg/kg body weight. Mice body weight and water consumption were adjusted weekly to adapt to the change of body weight and water consumption. After feeding on the Western diet for 3 months, the mice were killed and the atherosclerotic plaque formation was determined by Oil Red O staining. The ApoE−/− mice were killed by CO asphyxiation. Mouse aortas were dissected in cold PBS and cut open to expose the atherosclerotic plaques. After fixation in 4% formaldehyde for 16 h at 4 °C, the tissues were first rinsed in water for 10 min and then in 60% isopropanol. The aortas were stained with Oil Red O for 15 min with gentle shaking, and rinsed again in 60% isopropanol and then in water for three rinses. The samples were fixed on the cover slides with the endothelial surface facing upwards. The images were recorded using an HP Scanjet G4050. The plaque areas were determined using National Institutes of Health ImageJ software and calculated by expressing the plaque area relative to the total vascular area. The experiments were approved by the Hospital Human Subjects Review Committee (IRB approval number TSGHIRB 2-103-05-132) of Tri-Service General Hospital in Taipei and were conducted under the guidelines established by the Ethics Review Board of National Health Research Institutes, Taiwan. Written informed consent was obtained from all individuals. Human aortic tissue specimens were from patients with acute type-A aortic dissection. These samples were collected during emergency aortic surgery. The diseased segments of aorta (that is, dissecting aortic aneurysm) in these patients were all resected and replaced by an artificial inter-position graft. Specimens were fixed in paraformaldehyde, paraffin-embedded and cut into 5 μm sections. YAP Ser127 phosphorylation was determined by immunofluorescence imaging. HUVECs were transfected with pWCXIH-Flag-YAP-S127A (a gift from K. Guan, Addgene 33092) and 3× Flag pCMV5-TOPO TAZ (S89A) (a gift from J. Wrana, Addgene 24815) or pEGFP-N1 by Neon transfection system (Invitrogen, USA)32, 33. Four hours after transfection, cells were harvested and RNA was extracted using RNeasy Mini Kit (Qiagen, Germany). The extracted RNA samples were sent to Beijing Genomics Institute (BGI) for RNA-sequencing analysis. P < 0.05 and fold change >1.5 was used as a threshold for different regulated genes. DAVID tools were used for the pathways enrichment analysis and GlueGo was used for the Gene Ontology analysis. Ibidi flow system (IBIDI, Germany) was used to generate USS and disturbed flow (12 dyn cm−2 for USS and 0.5 ± 6 dyn cm−2, 1 Hz for disturbed flow). μ-slide I 0.4 Luers (IBIDI, LLC) was used for immunofluorescence studies. The slide was coated with 50 μg/ml fibronectin for 24 h. Seven thousand HUVECs were seeded onto the slide. After cells were adapted to medium containing 2% FBS (10% fatty acid free BSA for disturbed flow) for 6 h, the slides were mounted onto the Ibidi flow system. For immunostaining of USS-induced YAP/TAZ nuclear exportation, cells were subjected to USS for 6 h. For western blotting and reverse transcription real-time PCR analysis, the μ-slides were replaced with a custom-built flow chamber, which could accommodate more cells. Glass slides (75 mm × 38 mm; Corning) were coated with fibronectin (50 μg/ml). HUVECs were seeded on slides and allowed to attach on the bottom for 16 h. For USS, the medium was replaced with EGM supplemented with 2% FBS for 6 h. For disturbed flow, cells were incubated in EGM supplemented with 10% fatty-acid-free BSA (Sigma). The slides were mounted onto the flow chamber and connected to the Ibidi flow system. The cells were then subjected to USS or disturbed flow. For USS-induced YAP phosphorylation, 15 min of shear force was applied unless otherwise noted. For USS-induced YAP translocation, 6 h of shear force was applied. For reverse transcription real-time PCR analysis, 4 h of shear stress was sufficient to inhibit the expression of YAP/TAZ target genes. For reporter gene assay, 48 h of shear forces were applied to HUVECs. To construct the reporter plasmids for adhesion molecules, human genomic DNA was purified from HUVECs using a Universal Genomic DNA Extraction Kit Ver 3.0 (Takara, Japan). The promoters of ICAM1, SELE, CCL2 and CXCL1 were PCR amplified from human genomic DNA using the primers listed in Supplementary Table 1. A 2.1 kb fragment (−1784 to +328) from the ICAM1 promoter, a 2.2 kb fragment (−1807 to +475) from the SELE promoter, a 4 kb fragment (−3992 to +73) from CCL2 promoter and a 1.3 kb fragment (−1256 to +84) from CXCL1 promoter were amplified. The PCR products were gel purified by gel extraction kit (Takara, Japan) and digested with restriction enzymes. The digested fragments were gel purified and ligated to pGL3 reporter plasmid digested by corresponding restriction enzymes. The ligation products were then heat inactivated at 65 °C for 15 min and transformed into the DH5α competent cells. The Pro32Pro33 integrin was derived from pcDNA3.1-beta-3 (a gift from T. Springer, Addgene plasmid 27289) by point mutation34. Primers used for plasmids construction were included in Supplementary Table 1. To generate the adenovirus shuttle vector pShuttle-U6, the U6 promoter and 1.9 kb stuffer sequence was excised from pLKO.1 (a gift from D. Root, Addgene plasmid 10878) with NotI/XhoI and ligated into pShuttle plasmid pre-digested with restriction enzymes accordingly. Short hairpin RNA targeting mouse Taz was generated using a protocol similar to pLKO.1 shRNA plasmids (Addgene) construction protocol. Taz shRNA sequence, TRCN0000095951, which was validated by Mission shRNA (Sigma Aldrich), was used to generate shuttle plasmids for Taz shRNA. Recombinant adenovirus was generated using the AdEasy system35. Briefly, pShuttle-U6 vector containing shRNA was digested with PmeI and co-transformed with adenoviral backbone plasmid pAdEasy-1 for homologous recombination in E. coli BJ5183 cells. Positive recombinants were linearized by PacI digestion and transfected into HEK-293A cells for virus packaging. The medium and cells were collected until the cytopathic effect was apparent. After three cycles of freeze and thaw to release the virus, the cell debris was removed by centrifugation at 3,000 r.p.m. for 15 min. The virus-containing supernatant was collected by PEG precipitation, followed by dialysis against saline with 100K MWCO dialysis tubing (Spectrum Labs). Lentiviral shuttle plasmids for YAP (TRCN0000300325), TAZ (TRCN0000370007), Gα (TRCN0000036885) and ITGB3 (TRCN0000003236) shRNA were purchased from Sigma. Plasmid cocktail containing 1 μg of resultant shuttle plasmid, 750 ng of psPAX2 packaging plasmid and 250 ng of pMD2.G envelope plasmid were co-transfected to HEK-293FT cells. The medium was changed 15 h after transfection; 48 and 72 h after transfection, the medium containing the lentiviral particles was harvested then passed through 0.45 μm filters to remove cell debris. The virus was precipitated with PEG and suspended in PBS containing 4% sucrose. The lentiviral solutions were then aliquoted to vials and stored at −80 °C. YAP1 S127A was amplified from pWCXIH-Flag-YAP-S127A (a gift from K. Guan, Addgene 33092) and ligated to pAAV-MCS (Stratagene) to generate the pAAV-YAP1 S127A shuttle plasmid. A similar strategy was used to generate the pAAV-TAZ S89A from 3× Flag pCMV5-TOPO TAZ (S89A) (a gift from J. Wrana, Addgene 24815). pX601-AAV-CMV: NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA (a gift from F. Zhang, Addgene plasmid 61591) was used to generate the EC-specific Cas9 for Yap in vivo genome editing28. Three sgRNA sequences for Yap were predicted by CCTop (CRISPR/Cas9 target online predictor)36. ICAM2 endothelium-specific promoter from human was synthesized by GenScript and replaced the CMV promoter in pX601-AAV-CMV14. Primers used for sgRNA were included in Supplementary Table 1. The shuttle plasmids were co-transfected into HEK-293T with endothelial enhanced RGDLRVS-AAV9-cap plasmid (provided by O. J. Müller, Universität Heidelberg, Germany) and pHelper plamid (Stratagene)37. After co-transfection for 72 h, the AAV viral particles were isolated according to the protocol reported in ref. 38. Briefly, the cells were harvested and re-suspended in 1× restore buffer and the nuclei were extracted by homogenization. Viral particles were extracted by using nuclear lysis buffer. The viral particles were purified by PEG concentration, followed by dialysis against saline with 100K MWCO dialysis tubing (Spectrum Labs) to remove impurities, and concentrated. The viral titration was determined by qPCR and adjusted to 1010 plaque-forming units per ml in PBS containing 4% sucrose. For adenovirus-mediated Taz shRNA, viruses (109 plaque-forming units) were administered to ApoE−/− mice (male, 12 weeks old) that had been fed on Western diet (Research Diets) for 4 weeks, through tail vein injection. The mice were then fed on Western diet for 2 more months. The atherosclerotic plaque formation was visualized by Oil Red O staining. For AAV-mediated CA-YAP/TAZ overexpression and YAP-Cas9, the viruses (109 plaque-forming units) were administrated to ApoE−/− mice (male, 12 weeks old) through tail vein injection before feeding on Western diet or receiving the carotid partial ligation surgery. Statistics analyses were performed using GraphPad Prism 5.0. The sample sizes were not predetermined by statistical methods. The samples were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. At least three independent experiments were performed for all biochemical experiments and the representative images were shown. Results represent mean ± s.e.m. Student’s t-test (unpaired two-tailed) was used in the analysis. No samples, mice or data points were excluded from the reported analysis. Levels of probabilities less than 0.05 were regarded as significant. The RNA-seq data that support the findings of this study have been deposited in BioSamples database (https://www.ebi.ac.uk/biosamples/) under accession number SAMN04565728. All other data are available from the corresponding authors upon reasonable request.
News Article | November 20, 2016
One Doobert.com volunteer transported a three-year-old rescue dog from Jefferson, GA to his new foster home in Labadie, MO. Lincoln, NE, November 20, 2016 --( Volunteers found Jackie near Flat Rock Park in Brownwood, TX as a stray. They took her to the Corrine T. Smith Animal Center and listed her as a Boxer/Labrador Retriever because of the color of her coat. All Hounds on Deck from Lincoln, NE spotted a post about Jackie on All Hounds Rescue Network and recognized her as a Plott Hound. All Hounds on Deck rescues five specific breeds, and Plott Hound is one of them. Volunteers called on R.A.C.E., an organization that coordinates animal transports, to set up the 750-mile transport. They also set up a foster family in Nebraska to care for Jackie until her adoption. Thanks to the volunteers, All Hounds on Deck, R.A.C.E., and Doobert.com, Jackie, now named Sake by her foster family, gets another chance at a happy life. Volunteers use the custom-built software on Doobert.com to save animals by volunteering, fostering, and/or transporting animals. This software helps solve the most difficult aspect of coordinating animal rescues: transportation. With Doobert.com, animal lovers around the country come together to bring animals to their forever homes. Volunteers and organizations can sign up for free to rescue more animals at http://www.Doobert.com. Lincoln, NE, November 20, 2016 --( PR.com )-- Last week, Doobert.com and R.A.C.E. volunteers drove a 5-month-old Plott Hound named Jackie from Brownwood, TX to Lincoln, NE.Volunteers found Jackie near Flat Rock Park in Brownwood, TX as a stray. They took her to the Corrine T. Smith Animal Center and listed her as a Boxer/Labrador Retriever because of the color of her coat.All Hounds on Deck from Lincoln, NE spotted a post about Jackie on All Hounds Rescue Network and recognized her as a Plott Hound. All Hounds on Deck rescues five specific breeds, and Plott Hound is one of them.Volunteers called on R.A.C.E., an organization that coordinates animal transports, to set up the 750-mile transport. They also set up a foster family in Nebraska to care for Jackie until her adoption.Thanks to the volunteers, All Hounds on Deck, R.A.C.E., and Doobert.com, Jackie, now named Sake by her foster family, gets another chance at a happy life.Volunteers use the custom-built software on Doobert.com to save animals by volunteering, fostering, and/or transporting animals. This software helps solve the most difficult aspect of coordinating animal rescues: transportation. With Doobert.com, animal lovers around the country come together to bring animals to their forever homes.Volunteers and organizations can sign up for free to rescue more animals at http://www.Doobert.com. Click here to view the list of recent Press Releases from Doobert
Yu K.,University of Sichuan |
Yang J.,Hubei University of Medicine |
Jiang Y.,Animal Center |
Song R.,Chengdu BioSciTec Biotechnology Co. |
Lu Q.,University of Sichuan
Asian Pacific Journal of Cancer Prevention | Year: 2014
Background: Previous studies have investigated the association between the vitamin D receptor (VDR) BsmI polymorphism and colorectal cancer (CRC) susceptibility, but the results were conflicting. The aim of this study is to quantitatively summarize the relationship between this polymorphism and CRC risk. Materials and Methods: Two investigators independently searched the Medline, Embase, China National Knowledge Infrastructure (CNKI) and Chinese Biomedicine databases for studies published before November 2013. Summary odds ratios (ORs) and 95% confidence intervals (95%CIs) for VDR BsmI polymorphism and CRC were calculated in a fixed effects model (the Mantel-Haenszel method) and a random-effects model (the DerSimonian and Laird method) when appropriate. Results: This meta-analysis included 14 case-control studies, which included 10,822 CRC cases and 11,779 controls. Overall, the variant genotype (BB) of the BsmI was associated with a lower CRC risk when compared with the wild-type bb homozygote (OR=0.66, 95%CI: 0.49-0.88). Similarly, a decreased CRC risk was also found in the dominant and recessive models. When stratifying for ethnicity, source of controls, and study sample size, associations were observed among Caucasians, population-based studies and studies with large study sample size (>1000 subjects). Limiting the analysis to the studies within Hardy-Weinberg equilibrium, the results were persistent and robust. No publication bias was found in the present study. Conclusions: This updated meta-analysis suggests that the VDR BsmI polymorphism may be associated with a moderate protective effect against CRC.
Duan X.-H.,Fudan University |
Xu C.-Q.,Fudan University |
Huang J.-H.,Fudan University |
Zhou W.-J.,Animal Center |
Sun B.,Fudan University
Pharmaceutical Biology | Year: 2013
Context: Homocysteine-induced endothelial cellular senescence may contribute to some cardiovascular disorders. Icariin (ICA), a flavonoid derived from Epimedium sagittatum Maxim. (Berberidaceae), has been reported to increase production of nitric oxide (NO) and reduce reactive oxygen species (ROS) levels in human umbilical vein endothelial cells (HUVECs). Objective: To observe the effects of ICA on homocysteine-induced senescence and the underlying mechanisms in HUVECs. Materials and methods: ICA at concentrations of 0.1, 1, and 5 μM was added into homocysteine pretreated HUVECs. Cellular senescence was assayed by senescence-associated β-galactosidase (SA-β-gal) staining and cumulative population doublings (CPDs). ICA (5 μM) was given orally to homocysteine-treated rats, luminal surface of aortic artery of rats was subjected to SA-β-gal staining. Protein expression was measured by western blot. Results: Homocysteine significantly increased cellular senescence both in vitro and in vivo. After treatment by ICA, the percentage of SA-β-gal-positive cells, and the ROS level significantly decreased. The CPDs were partially restored. ICA also significantly reduced the mean density of SA-β-gal staining in vivo. We found that NO production and phosphorylation of AKT, ERK, and endothelial NO synthase (eNOS) were elevated by ICA in HUVECs. Furthermore, the increased level of NO production was fully abolished by the phosphatidylinositol-3-kinase (PI3K) inhibitor wortmannin. The mitogen-activated protein kinase (MEK) inhibitor PD98059, which can inhibit phosphorylation of ERK, did not show this ability. Discussion and conclusion: Our results indicate that ICA delays homocyteine-induced endothelial senescence in vitro and in vivo. Activation of PI3K/Akt-eNOS-dependent signaling pathway may be responsible for this efficacy of ICA. © 2013 Informa Healthcare USA, Inc.
News Article | November 30, 2016
Help make the holidays brighter for the shelter animals by making a donation to BrightSide Animal Center. The holiday pet drive, hosted by Premier Printing Solutions is collecting much-needed items to benefit BrightSide Animal Center in Redmond, Oregon.