text:
Phase I Study of Cellular Immunotherapy for Recurrent/Refractory Malignant Glioma Using Intratumoral Infusions of GRm13Z40-2, An Allogeneic CD8+ Cytolitic T-Cell Line Genetically Modified to Express the IL 13-Zetakine and HyTK and to be Resistant to Glucocorticoids, in Combination With Interleukin-2
|The safety and scientific validity of this study is the responsibility of the study sponsor and investigators. Listing a study does not mean it has been evaluated by the U.S. Federal Government. Read our disclaimer for details.
|ClinicalTrials.gov Identifier: NCT01082926
Recruitment Status : Completed
First Posted : March 9, 2010
Last Update Posted : June 8, 2015
- Study Details
- Tabular View
- No Results Posted
- Disclaimer
- How to Read a Study Record
RATIONALE: Biological therapies, such as cellular adoptive immunotherapy, may stimulate the immune system in different ways and stop tumor cells from growing. Donor T cells that are treated in the laboratory may be effective treatment for malignant glioma. Aldesleukin may stimulate the white blood cells to kill tumor cells. Combining different types of biological therapies may kill more tumor cells.
PURPOSE: This phase I trial is studying the side effects and best way to give therapeutic donor lymphocytes together with aldesleukin in treating patients with stage III or stage IV malignant glioma.
|Condition or disease
|Anaplastic Astrocytoma Anaplastic Ependymoma Anaplastic Meningioma Anaplastic Oligodendroglioma Brain Stem Glioma Ependymoblastoma Giant Cell Glioblastoma Glioblastoma Gliosarcoma Grade III Meningioma Meningeal Hemangiopericytoma Mixed Glioma Pineal Gland Astrocytoma Brain Tumor
|Biological: therapeutic allogeneic lymphocytes Biological: aldesleukin Other: laboratory biomarker analysis Procedure: positron emission tomography
|Phase 1
I. To assess the safety of GRm13Z40-2 CTL CNS loco-regional cellular immunotherapy in research participants with recurrent or refractory/ progressive malignant glioma (WHO Grades 3 or 4).
II. To assess the safety of convection enhanced delivery (CED) of recombinant human Interleukin-2 (rhuIL-2) used in conjunction with GRm13Z40-2 CTL adoptive transfer.
I. To investigate the ability of 9-(4-fluoro-3-hydroxy-methyl-butyl) guanine (18FHBG) positron emission tomography PET to image GRm13Z40-2 CTL's in research participants.
II. To study the impact of concurrent dexamethasone administration on the tempo and magnitude of T cell allograft rejection responses in treated research participants by tracking the frequency of anti-GRm13Z40-2 immune responses in serially acquired peripheral blood samples.
III. To evaluate ganciclovir administration for ablating transferred GRm13Z40-2 in vivo should significant graft-mediated toxicities be encountered.
OUTLINE: Patients receive GRm13Z40-2 therapeutic allogeneic lymphocytes intratumorally (IT) over 10 minutes on days 1 and 3 and aldesleukin IT over 3 hours on days 2-5 (days 1-5 in week 2). Treatment repeats every week for 2 courses in the absence of disease progression or unacceptable toxicity.
After completion of study treatment, patients are followed annually for at least 15 years.
|Study Type :
|Interventional (Clinical Trial)
|Actual Enrollment :
|6 participants
|Intervention Model:
|Single Group Assignment
|None (Open Label)
|Primary Purpose:
|Official Title:
|Phase I Study of Cellular Immunotherapy for Recurrent/Refractory Malignant Glioma Using Intratumoral Infusions of GRm13Z40-2, An Allogeneic CD8+ Cytolitic T-Cell Line Genetically Modified to Express the IL 13-Zetakine and HyTK and to be Resistant to Glucocorticoids, in Combination With Interleukin-2
|Study Start Date :
|May 2010
|Actual Primary Completion Date :
|September 2013
|Actual Study Completion Date :
|September 2013
Experimental: Arm I
Patients receive intratumoral GRm13Z40-2 therapeutic allogeneic lymphocytes over 10 minutes on days 1 and 3 and intratumoral aldesleukin over 3 hours on days 2-5 (days 1-5 in week 2). Treatment repeats every week for 2 courses in the absence of disease progression or unacceptable toxicity.
Biological: therapeutic allogeneic lymphocytes
Given intratumorally
Other Name: ALLOLYMPH
Biological: aldesleukin
Given intratumorally
Other Names:
Other: laboratory biomarker analysis
Optional correlative studies
Procedure: positron emission tomography
Optional correlative studies
Other Names:
- Safety of GRm13Z40-2 CTL CNS loco-regional cellular immunotherapy [ Time Frame: Daily for first 2 weeks, weekly for month 1, every other week for month 2 , monthly for 6 months ]
- Safety of convection enhanced delivery (CED) of recombinant human Interleukin-2 (rhuIL-2) used in conjunction with GRm13Z40-2 CTL adoptive transfer [ Time Frame: Weeks 1 and 2 ]
- Toxicity as assessed by NCI CTCAE version 4.0 [ Time Frame: During treatment and up to 21 days after the last GRm13Z40-2 or CED rhuIL-2 infusion ]
- Ability of 9-(4-fluoro-3-hydroxy-methyl-butyl) guanine (18FHBG) positron emission tomography PET to image GRm13Z40-2 CTLs [ Time Frame: Prior to immunotherapy and 3 weeks post immunotherapy ]
- Impact of concurrent dexamethasone on the tempo and magnitude of T cell allograft rejection responses by tracking the frequency of anti-GRm13Z40-2 immune responses in serially acquired peripheral blood samples [ Time Frame: Post infusion day 1, weeks 2-4 and week 8 ]
- Evaluation of ganciclovir administration for ablating transferred GRm13Z40-2 in vivo should significant graft-mediated toxicities be encountered [ Time Frame: When/if grade 3 or 4 toxicity occurs ]
Choosing to participate in a study is an important personal decision. Talk with your doctor and family members or friends about deciding to join a study. To learn more about this study, you or your doctor may contact the study research staff using the contacts provided below. For general information, Learn About Clinical Studies.
|Ages Eligible for Study:
|18 Years to 70 Years (Adult, Older Adult)
|Sexes Eligible for Study:
|Accepts Healthy Volunteers:
Inclusion Criteria:
- Histological verification of grade III or IV MG at original diagnosis
- Radiographic evidence of progression/recurrence of the measurable disease more than 12 weeks after the end of radiation therapy
- Expression of IL13Ralpha2 by immunohistochemistry
- Karnofsky performance status (KPS) >= 60
Disease recurrence/progression in the cerebral hemisphere, which is in at least one area of enhancement amenable to biopsy after protocol enrollment in the following locations:
- Adjacent or near previous resection cavity
- Distant from primary location; this includes tumor spread to contralateral hemisphere, corpus callosum, thalamus, basal ganglion, or subependymal locations
- Research participant has recovered from toxicity of prior therapies; an interval of at least 12 weeks must have elapsed since the completion of radiation therapy; at least 6 weeks since the completion of a nitrosourea-containing chemotherapy regimen; and at least 4 weeks since the completion of a non-nitrosourea-containing cytotoxic chemotherapy regimen; if a patient's most recent treatment was with a targeted agent only, and s/he has recovered from any toxicity of this targeted agent, then a waiting period of only 2 weeks is needed from the last dose and the start of study treatment, with the exception of bevacizumab where a wash out period of at least 4 weeks is required before starting study treatment
- History of prior treatment with Temodar if no evidence of intolerance; documentation of intolerance to Temodar is not required
- Creatinine < 1.6
- White blood cell (WBC) >= 2,000/dl (or absolute neutrophil count [ANC] > 1,000) Platelets >= 100,000/dl unsupported by transfusion or growth factor, international normalized ratio (INR) < 1.3
- Bilirubin < 1.5
- Serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) < 2 X upper limits of normal
- Female research participants of childbearing potential must not be pregnant as evidenced by a serum beta-HCG pregnancy test obtained within 7 days of enrollment
- Research participants having reproductive potential must agree to use effective contraception during participation on this protocol
- In the opinion of the neurosurgeon, research participant requires on-going dexamethasone therapy
Exclusion Criteria:
- Survival expectation less than 4 weeks
- Pulmonary- Requirement for supplemental oxygen use that is not expected to resolve within 2 weeks, Cardiac- Uncontrolled cardiac arrhythmia, hypotension requiring pressor support, Renal- Dialysis dependent, Neurologic- refractory seizure disorder, clinically evident progressive encephalopathy
Tumors with the following characteristics:
- Large tumor recurrence causing significant symptoms from brain shift or mass effect, and thus not requiring "decompressive" craniotomy
- Tumors located primarily in the basal ganglion or thalamus
- Tumors with significant involvement of midbrain, cerebellum, pons and medulla will be excluded due to neurological risks associated with edema exacerbation from therapy
- Research participants with any non-malignant intercurrent illness which is either poorly controlled with currently available treatment, or which is of such severity that the investigators deem it unwise to enter the research participant on protocol shall be ineligible
- Positive human immunodeficiency virus (HIV) serology based on testing within 4 weeks of enrollment
- Research participants being treated for severe infection or who are recovering from major surgery are ineligible until recovery is deemed complete by the investigator
- Failure to understand the basic elements of the protocol and/or the risks/benefits of participating in this pilot study
- History of ganciclovir and/or magnetic resonance imaging (MRI) contrast allergy or intolerance History of intolerance to IL-2
To learn more about this study, you or your doctor may contact the study research staff using the contact information provided by the sponsor.
Please refer to this study by its ClinicalTrials.gov identifier (NCT number): NCT01082926
|United States, California
|City of Hope
|Duarte, California, United States, 91010
|Principal Investigator:
|Behnam Badie
|City of Hope Medical Center
|Responsible Party:
|City of Hope Medical Center
|ClinicalTrials.gov Identifier:
|Other Study ID Numbers:
|First Posted:
|March 9, 2010 Key Record Dates
|Last Update Posted:
|June 8, 2015
|Last Verified:
|June 2015
adult anaplastic astrocytoma
adult anaplastic ependymoma
adult anaplastic meningioma
adult anaplastic oligodendroglioma
adult brain stem glioma
adult ependymoblastoma
adult giant cell glioblastoma
adult glioblastoma
adult gliosarcoma
adult grade III meningioma
adult meningeal hemangiopericytoma
adult mixed glioma
adult pineal gland astrocytoma
recurrent adult brain tumor
Los Angeles
Brain Neoplasms
Neuroectodermal Tumors, Primitive
Neoplasms, Neuroepithelial
Neuroectodermal Tumors
Neoplasms, Germ Cell and Embryonal
Neoplasms by Histologic Type
Neoplasms, Glandular and Epithelial
Neoplasms, Nerve Tissue
Central Nervous System Neoplasms
Nervous System Neoplasms
Neoplasms by Site
Brain Diseases
Central Nervous System Diseases
Nervous System Diseases
Neoplasms, Vascular Tissue
Meningeal Neoplasms
Antineoplastic Agents
Analgesics, Non-Narcotic


text:
06 | Mar | 2016
RyR Trade Press Trip
February 29- March 6, 2016 - Attendee Backgrounders
|Social Media
|The Bristol
Charles Ford
|Sommelier/Brand Ambassador
294 Twitter Followers
712 Instagram Followers
|He began his career as a line cook at Jackie Shen's Red Light Restaurant. In October 2011, after over a year at Everest, Charles followed Dave to The Bristol, where he joined the team as Assistant Manager. While continuing to find his niche in the hospitality industry, Charles became a Certified Sommelier with Court of Master Sommeliers. He is currently the Assistant General Manager and Wine Director, fulfilling his passion for creating memorable experiences for guests through both great service and exceptional wine.
|Wine-all The Time
Marissa A. Ross
|Tastemaker and Wine Reporter
18.1K Instagram Followers
12.9K Twitter Followers
|Marissa is a leisure enthusiast, writer and wine drinker. In 2015, Marissa won both Editor's Choice and Reader's Choice awards at Saveur Magazine's Blog Awards, and was also named one of Wine Enthusiast's 40 Under 40. She is a contributor for VICE Munchies and the author of UNWINED (Plume, 2017). She recently started contributing to Bon Appetit print and online editions through summer 2016.
|Southern Wine & Spirits
Dana Gaiser
|Wine Import and Budding Sommelier
710 Instagram Followers
681 Twitter Followers
|A 14-year veteran of the wine industry, Dana has served as a Sommelier and Beverage Director at top restaurants such as Veritas, Estiatorio Milos and others in New York, Nantucket and California. He is currently Key Accounts Director for Lauber Imports, the fine wine division of the largest wine and spirits distributor in the U.S.
|Vine Pair
Emily Bell
6,269 Twitter Followers
37,598 Facebook Followers
6,644 Instagram Followers
|Emily has been writing about food, drink, and the culture and politics thereof for several years now - despite all of which, she is still weirdly hungry and thirsty.
|Eddie V's Restaurants
Brian Phillips
|Advanced Sommelier
282 Twitter Followers
|Brian is an Operating Partner for Eddie V's Prime Seafood, an Advanced Sommelier, a Certified Wine Educator and a Certified Food & Beverage Executive
|FWx Food & Wine
Ethan Fixell
20.5K Twitter Followers
152 Facebook Followers
1,859 Instagram Followers
|Ethan is a a Certified Cicerone® and frequently writes about beer and spirits for Food & Wine's FWx, Tasting Table, Food Republic, and Quartz. Additionally, he has contributed to Food & Wine, Vanity Fair, Travel + Leisure, Playboy, Saveur, DuJour, and Draft Magazine, and previously wrote a weekly column for Serious Eats called "On the Beer Trail."
|Bar Boulud
Jennifer Foucher
|Sommelier / Sommelier Challenge International Judge
536 Twitter Followers
321 Instagram Followers
|Jennifer is a certified sommelier at Bar Boulud (formerly the wine director at Del Posto) and Sommelier Challenge International Judge. She will be participating in the 2016 Rieslingfeier this month.
|Fred Dexheimer
Fred Dexheimer
|Master Sommelier/Wine Consultant
7,149 Twitter Followers
1,112 Instagram Followers
|Fred Dex is one of only 210 Master Sommeliers in the world and the only one holding that honor in the Carolinas. Fred's career as a wine and beverage professional spans nearly two decades and has touched upon nearly every aspect of the business.
|Drink Me
David Furer
|Wine Consultant
35K Twitter Followers
17K Facebook Followers
|Since 1986 David has performed many roles in the alcohol beverage industry–lecturer and teacher to both pros and consumers, consultant to numerous wine regions and countries, event organizer and host, retail buyer, sommelier and restaurant manager, and wine/spirits/beer writer/author/judge. In addition to holding an Advanced level certification from the Court of Master Sommeliers and attaining Certified Wine Educator from the Society of Wine Educators, he's worked at five German wineries. His lecturing skills have been appreciated at the University of Chicago and, while residing in England, at Oxford and Cambridge universities
|Wine on the Road
Ben Weinberg
|Writer/Wine Consultant
1,265 Twitter Followers
51 Facebook Followers
|Ben is a member of the American Society of Journalists and Authors and travels extensively in Italy, France, Spain, Germany, Argentina, Chile, Washington, Idaho and Colorado. He regularly attends wine- and writing-focused events such as the Symposium for Professional Wine Writers at Meadowood Napa Valley. He has been the Rocky Mountain Editor for The Tasting Panel Magazine and has written for The World of Fine Wine, Wine Enthusiast, Wines & Vines, Wine Country International, Nation's Restaurant News, Worth Magazine, Where Magazine, Denver Magazine, and The Colorado Statesman. Ben also authors the Wine Roads travel ebook series in conjunction with Board and Bench (formerly the Wine Appreciation Guild).
|Conde Nast Traveler
Krisanne Fordham
|Freelance Travel + Wine Writer
2,153 Instagram Followers
1,370 Twitter Followers
|Krisanne is a travel, culture and wine writer for Condé Nast Traveler, CNN Travel, Departures, and Travel + Leisure. Previously, she was a staff editor at Condé Nast Traveler and The AOL-Huffington Post Media Group. Krisanne grew up in Sydney, Australia and splits her time between Brooklyn, New York and Umbria, Italy.
RyR Trade + Press Trip February 29- March 6, 2016 Attendee Backgrounders

subset: commoncrawl


text:
Tahoura Samad 15:18, October 15 2010 (EDT)
-went down to Koshland to do confocal with Scott from the king lab. Could only load 30 ul of sample at time. Looked at NLS and 51 after induction fifteen minutes after feeding. -saw one possible hit, but set up wasn't optimal -choanos were suspended in low melt temp agarose. -probably better to use scope in Stanley. -lysis looks like it occured in pmll KC. miniprepped the plasmid, but probably will not use because it took so long. Also streaked out control, and will retest for lysis -for the future: JTK030 and 159 have different copy numbers. Probably why timing is different for 159 with 2801 and NLS which is 030. -New lifeact part 171 in 1600 still did not grow up
Tahoura Samad 15:18, October 14 2010 (EDT)
-started fresh choano culture by adding Kan to choano media culture. -picked colonies of AKS NLS with 51 and 52 as well as 2801 with 51 and 52 to look at under Koshland confocal. -checked on colonies of iGEM171 in 1600 (p15 spec). Did not grow up. Talk to Tim. -tested lysis of PCR of 51 digested and ligated in pmll KC. Did not appear to have lysed after 6 hours. kept in shaker
Tahoura Samad 15:18, October 13 2010 (EDT)
-set up test with NLS in 030 for 51 and 52 for confocal in upstairs stanley. -52 still behaving oddly, with little hits/fluoresence -got an excellent image of 51 at timepoint -15. showed fluoresence clearly filling a choano shaped object. YIPEEEEEE!!! -controls for new 11 cells looked clean. Transformations grew up alright. -controls for Daniela's 1600 payload cells looked clean.
Tahoura Samad 15:18, October 12 2010 (EDT)
-made competent cells of 11 and transformed them with 51 and 52 ran controls on AC, S, K.
Tahoura Samad 15:18, October 11 2010
-restransformed 51 . 52 into 9.
Tahoura Samad 15:18, 6 October 2010 (EDT)
-checked lysis Tecan with Daniela for iGEM171 with 51 and 52 in 1256. Appears to have lysed, just slowly. Tim is E/Bing it into p15a vector
Tahoura Samad 15:18, 5 October 2010 (EDT)
-fed choanos
Tahoura Samad 15:18, 4 October 2010 (EDT)
-did choano assays with 1934 NLS in JTK030 at induction time points +30, +15, 0, -15. -Had dones more hits in 51 for 0 and -15 time points. could barely move in field of view without seeing a hit! -52 did not have any hits at all.
Tahoura Samad 15:18, 24 September 2010 (EDT)
- Started Eco/Bam of stuff into CK.
- Mass production of choanos.
Tahoura Samad 18:36, Sept 23 2010 (EDT)
- Ran assay with 51 and 52 in '8', which is Bjh1255 in JTK030, and '9' which is Bjh1934 in JTK030. Also tried to run Conor's 52 with iGEM159.
- Didn't see invitro lysis for anything in 8, or with Conor's new lifeact construct.
- Will transform over weekend so Tim can rerun assay on Wednesday.
To Do
- Start mass production of choanos to run Josh's plasmids and Tera's plasmids.
- Test lysis of Amy's CK stuff
Tahoura Samad 18:36, Sept 22 2010 (EDT)
- Ran lysis of Amy's Stuff (51 and 52) which did not lyse after 2 hours
- Tried to do lysis at room temperature. Induce at 11 am, and did not see lysis at 6 pm. Looks like we may have had lysis by the next day at around 10 am.
- picked stuff for choano assays again tomorrow. 4 colonies each because of poor growth seen on Monday.
Tahoura Samad 18:36, Sept 21 2010 (EDT)
- Choano care
- ReKan'd a couple plates to get rid of biofilms.
Tahoura Samad 18:36, Sept 20 2010 (EDT)
- Daniela accidently induced same sample twice, so scrapped '8' and '9' assay.
- Ran choano assay on just 1968 in '11' with plain '11' as control. Didn't really see any of the same successful choano phenotype after 3 hours after feed at 90,60 and 30 min induction points. In addition, adding more bacteria (5ul vs 1ul) doesn't really appear to help efficiency. Just see a lot of stuff lysing outside of choanos. May not be limiting factor.
Tahoura Samad 18:36, Sept 19 2010 (EDT)
- picked stuff for choano assay on Monday which includes 51 and 52 in 8 and 9, as well as Bjh1968, just self-lysis.
Tahoura Samad 18:36, Sept 18 2010 (EDT)
- played with new iGEM friends with Christoph.
- Out of five, only one, the Tetrahymena, appeared to have eaten the bacteria within the 4 hours we looked at them. At the final hour, samples were sluggish and swimming in erratic circles=not healthy.
- Concerned about size being too big to see lysis even if it was occuring.
Tahoura Samad 18:36, Sept 17 2010 (EDT)
- slept through self-lysis assay induction points. Will run on Sat or Monday.
Tahoura Samad 18:36, Sept 14 2010 (EDT)
- ran same choano assay again. SUCESSS!!! Details in spreasheet. :D
Induction timepoints:
Tahoura Samad 18:36, Sept 10 2010 (EDT)
- Look at choanos again after about 24 hours. FP signal is reallly low, takes about 15000 ms to really pick up anything in lysed samples. Not a lot of bacteria anywhere, especially in lysed samples.
- Had Christoph transform again.
Tahoura Samad 18:36, Sept 9 2010 (EDT)
- Ran choano assay. Induction starting at 10:15, with time points at 90,75,60,45,30,15. Fed at 12:00, So block was in shaker 15 minutes longer than expected. Started looking at approximately 3 pm.
- GFP=300ms
Phase=162 ms
- SUCCESS!!!!!! Saw green choanos in 51 at 90, 75 time points. Other samples were photobleached. Will save samples to look at again tomorrow and see if FPs last.
Tahoura Samad 18:36, Sept 8 2010 (EDT)
- picked colonies to assay tomorrow. Grew up in TB. Will aspirate off ASW, and then resuspend in choano media.
Tahoura Samad 18:36, Sept 7 2010 (EDT)
- Set up TECAN with LB, LB culture resuspended in ASW, Choano, 50:50, 25:75, 75:25 LB:ASW, and same for TB, TB resuspended.... S
- Graphs show clear lysis for everything except for ASW and 50:50 for both 51 and 52.
Tahoura Samad 18:36, Sept 6 2010 (EDT)
- Christoph ran lysis assays over weekend in LB/ASW and choano media. Said he saw lysis in all of them. Will run Tecan tomorrow to confirm.
- Did microscopy assay of 51 and 52 induced 90, 75, 60,45,30,15 mins ahead of time after 24 hours. Did not see green phenotype anywhere, or really all that much green. Not a success, but not surprising considering that we don't really see lysis in ASW.
Tahoura Samad 18:36, Sept 3 2010 (EDT)
- Microscopy comparing Tb vs LB 1, 2, 5, 10 ul.
- Ran PCR of Pcons with Lifeact FP.
- Gave payload project to conor.
- Ran quick macroscale assay of lysis in ASW. Not apparent after four hours.
Tahoura Samad 18:36, Sept 2 2010 (EDT)
- Had Christoph induce cells, started assaying at 3:00. Fatal error. Not enough bacteria for cells to eat. Will do a concentration microscopy test tomorrow to confirm.
Tahoura Samad 18:36, Sept 1 2010 (EDT)
- sequencing results for 7 looked good.
- picked colonies to feed and assay on Thurs.
- ran lysis assay in LB for 51, and 52. Saw clear lysis after about 2 hours. 52 seems to lyse a little sooner then 51.
- got two Pcon's from Tim/Sushant to put on the front of Lifeact. PCR off them with ca998 and g01001 and then Eco/Bam Eco/Bgl. Tape samples to the inside of TIm's fridge when done. WIll do tomorrow.
Tahoura Samad 18:36, 31 August 2010 (EDT)
- miniprepped 3, 7, 8, and sent 7 in for sequencing with ca998.
- controls for "11" cells looked good
- picked cells to run preliminary lysis assay on Weds. before thursday
Tahoura Samad 18:36, 30 August 2010 (EDT)
- Did colony 2K for Pbad. Lifeact GFP. Band should be around 2000. Will miniprep, 3, 7, 8 tomorrow.
- Transformed 51, and 52 into "11" and JTK159 to replenish supply of PDD.
- WIll assay on Wed, Thurs?
To Do
- Miniprep 3, 7, 8 from col2k
- pick colonies for assay on Wed. Either assay and invitro, or just invitro.
- Sequence pbad. lifeactGFP.
Tahoura Samad 19:29, 27 August 2010 (EDT)
- Made JTK2801 competent cells "11". Will run controls when I transform on Monday.
- Make sure to email David Chen two days in advance about assaying.
Tahoura Samad 19:29, 26 August 2010 (EDT)
- Analyzed Tecan data Tim. 51 and 52, along with 41 lysed. Will hopefully will be continuing lysis assays with 51, 52.
- Eco/Bam Eco/Bgl with Neb3 AraC. Pbad with Lifeact RFP.
- picked up JTK8021 to make comp cells with and assay.
- picked Bjh2252 to miniprep and assemble with AraC.Pbad.
To Do
- ReEcoBam 2271 into CA. First check with Tim though.
- Check with Tim about assaying when he's gone
- Check with Tim what the heck to do with AraC. Pbad LIfeact
- Make comp cells with JTK8021.
Tahoura Samad 19:29, 25 August 2010 (EDT)
- Ran lysis assay with 41, 42, 45-52 in JTK030, and in 159 3 different colonies. Will analyze Tecan Data tonight and tomorrow.
- Talked to Tim about Lifeact parts. Lifeact GFP sequenced well but is not green. Abandon for now. LifeactRFP is heavily mutated in the Lifeact Region. Abandon
- Will start assembly of Pbad. with LIfeact GFP and RFP. Grow up titration curve? Talk to Tim
Tahoura Samad 19:29, 23 August 2010 (EDT)
- 2ab assembly products were not green and red. Will ask Daniela to pick on Tuesday to miniprep and see what is going on.
- Neb2 Eco/Bgl LifeactRFP are now appearing red. Tim miniprepped them for me. Sent them in for sequencing to see what is going on.
- Talked to Daniela and Tim about end of lysis assay on Friday. (everything in JTK159, plated on both Kan and Amp. Looks like just 51 and 52 lysed.
- Retransformed 41, 42, 45-52 into Jtk159 and Jtk030. Prepped blocks for Daniela to pick tomorrow so I can assay on Weds.
- Picked BJh2541, Bjh2252 Eco/Bgl in Neb 2 and Bjh2251 by 2ab for Daniela to miniprep tomorrow.
- Also transformed Neb3 EcoBgl assembly products.
Tahoura Samad 19:29, 20 August 2010 (EDT)
- Ran lysis assay in TB for 41, 42, 45-52. Left early to move in and will get results from Daniela.
Tahoura Samad 16:35, 18 August 2010 (EDT)
New Payload Work
- Saw growth on plates, but no green and red colonies, so assembly was a failure.
- Redid shortcut method of assembly, but digested EcoBgl with NEB3 instead of NEB 2.
- Also created new assembly trees and Eco Bammed everything into pmll vectors.
- Retransformed 41, 42, 45-52 and plated on AC for 41 and 42 and AK for 45-52 to avoid contamination issues. Asked Daniella to pick them for me tomorrow. Daniela said she would pick colonies for EcoBam stuff and do 2ab assembly on Friday. I'll do lysis testing.
Tahoura Samad 16:35, 17 August 2010 (EDT)
New Payload Work
- transformed everything from yesterday
Biochipper's Device
- tried and failed at loading it. Maybe tape seal was no good?
- redid Amy's lysis assay in TB with no sucess. Amp plates seem to be contaminated and we plated on Amp. only. Will retransform 41, 42, 45-52 tomorrow.
Tahoura Samad 16:35, 16 August 2010 (EDT)
New Payload Work
- retried Christoph and Conor's shortcut for assembly of the two new payloads by PCRing of PCON from Josh (plate 5, well c10) with phusion. Expected band length was 1000 bp. Cut and gel purified, and digested with Eco Bam. Also digested Lifeact GFP and RFP from Jin with Eco Bgl. Ligated Pcon eco bam into Eco/bgled lifeact rfp and gfp and transformed into Pir Lefty.
Lysis Testing
- Amy ran lysis test and saw no lysis. Will rerun test for her in just TB tomorrow.
Tahoura Samad 16:35, 13 August 2010 (EDT
- split choanos
- made JTK159 cells.
Tahoura Samad 16:35, 12 August 2010 (EDT)
- Met up with Gayla and Tigran to learn how to make chips with Daniela. Practiced pouring PDMS, cutting devices, bonding etc. Make sure to get Gayla's guide.
- Found out that our jtk030 cells also have ptet in front of pir, which is leading to other problems. Tim grew up some JTK159, and we will make cells tomorrow.
Tahoura Samad 16:35, 11 August 2010 (EDT)
- found out that all our payloads have pTet, which means they are useless with TetR. Need to make two new payloads. Abandoned assembly with new iBB because it includes pTet in front of GFP.
- Helped Amy wash samples for self-lysis test without payload.
- Ran TECAN along with macro scale test. Saw lysis for several constructs! See excel sheet.
- Also did stickiness assay to confirm lysis.
Tahoura Samad 16:35, 10 August 2010 (EDT)
Tahoura Samad 9:25, 9 August 2010 (EDT)
- set up lysis assay. saw no lysis after 3 hours. grew upstairs in shaker.
- Choanos do not like to be left over the weekend.
- picked stuff for assembly final step.
Tahoura Samad 18:32, 8 August 2010 (EDT)
- picked colonies for lysis assay on Monday and grew up in TB
- tested 1934 comp cells by transforming with 47.
Tahoura Samad 11:25, 6 August 2010 (EDT)
Choano Assay
- Set up lysis assay for 47 and 48 with payload in LB vs in ASW, with Mc1061 with and without ATC as a control. Results were that both control and positive ATC OD dropped off at the same rate, possibly because the temperature control on the TECAN is at 26 degrees rather than 37.
- Lifeact RFP didn't grow up very well so was unable to get it to grow up well enough to make comp cells. Will retransform next week.
Tahoura Samad 11:25, 5 August 2010 (EDT)
Choano Assay
- growth of cells in LB was poor, which confirms that life-time on each transformation is less than a week. Ideally within the first four days of life of the plate. Set up chonao assay again but saw no lysis. Will redo in TECAN tomorrow using TB to get better growth.
- made competent cells for Lifeact GFP in JTK030 and Bjh1934 in JTK030.
- none of choano cultures, including 1:2 was at assaying density yet. Fed each 40 ul and let sit.
Tahoura Samad 9:32, 4 August 2010 (EDT)
Choano Assay
- Set up chonao assay. Washed saturated LB culture of 47 and 48 with payload delivery in choano media, ASW, TB and LB. Using 2 3 mL aliquots, added ATC to one tube and none to other to serve as control. DId not see lysis after 3 hours. Will grow up in LB and redo test on Thursday.
- re-cotransformed JTK030 with Bjh1934 and pBca1256 for assaying this week. Transformed JTK030 with lifeact GFP and RFP and 1934to make comp cells.
- Eco Bammed RFP Boo15 out of hybrid plasmid into KC, and transformed into MC1061 pir righty.
- Quintara resequenced Rbs ibb, gfp and it looks perfect. Tim deleted the first three bp off the oligo, so that's why there is that deletion in the front of the rbs.
- obtained a saturated choano culture. Split 1:2, 1:5, !;15 and fed 40 ul of choano smoothie. Will observe growth as compared to choanos in choano media.
Tahoura Samad 13:32, 3 August 2010 (EDT)
Choano Assay
- ran assay of taking pictures of choanos eating just payload bacteria every five minutes for an hour. Saw photobleaching at a round minute 82. Arrived at scope 15 minutes after having fed choanos and were not seeing any rod phenotype. Hard to conclude the details of choano digestion because choanos keep moving slightly in and out of plain, messing up focus of FPs. Need to figure out how to fix choanos in space.
- miniprepped Rfp terminator and GFP rbs ibb. RFP terminator mapped strangely. Its a hybrid plasmid. Solution: PCR off hybrid plamsid using Ca998, G0100, digest, and ligate into correct plasmid.
Lysis Assay
- Did a loose assay to check if construct 47 and 48 without payload were lysing in ASW. Grew up in LB to saturation, washed and added ATC. Did not lyse. Picked and grew up colonies in TB to redo assay tomorrow. Will use just TB culture as a control, because it should lyse obviously within two hours according to Amy.
Tahoura Samad 16:03, 2 August 2010 (EDT)
Chonao Assay
- Checked 3 day culture of flavobacterium free chonaos. Not as dense as a 3 day culture should be.
- Ran assay of TetR 47, TetR48 at +15, 0 -15 and -30. After 1 hour, seeing some rod shaped bacteria, but some empty choanos. Didn't see desired phenotype after 1 or 4 hours. Images are clearer without biofilms though.
- Surprised not to see some of landmarks of digestion we expected to see. Rounding up? Run 1 hour assay tomorrow to establish baseline
- streaked out JTK030. Pick to make comp. cells tomorrow. Make iGEM glycerol stock of Jtk030.
- picked LIfeact GFP to make comp cells today. In JTK030.
- picked LIfeact RFP in 1256
Tahoura Samad 16:03, 29 July 2010 (EDT)
Choano Assay
- began set up for choano assay at 10:00.
- used whole mLs for each of three hour time points because of loss of choanos during washing.
- 10:15 am, made 3 1 ml aliquots of each TetR 47 and TetR 48. TetR 47 did not grow up as well as TetR 48.
- 10:30 added ATC to samples with ATC for 90 minutes. (1 μL per mL of bacteria, keep in shaker.)
- 10:45 split choanos 1:15.
- 11:00 Added ATC to samples with ATC for 60 minutes. Started filtering choanos
- 11:30 Added ATC to samples with ATC for 30 minutes.
- 11:40 Added NH4CL to samples that needed it.
- 12:00 Fed choanos. Took about 10 minutes.
- 12:35 Washed choanos with 1 mL of choano media and chased with 25 mL of white ecoli (plain MG1655)
- 1:00 Started filtering choanos
- looked at chonaos at around the two hour mark. Very little fluorescence at all, only getting fluoresence when we bumped up the exposure to like 500 ms. Possible that the cells are lysing way before they are eaten. Chris says to try intervals between 15 minutes before to 15 minutes after feeding.
- in assay, chase with less white ecoli.
- from assay, can conclude that choanos complete digestion definitely within 2 hours, probably within 1.
- no such thing as narchoanos. :( just choanos with two flagella.
IBB Work
- miniprepped 3 of 4 colonies that weren't cotransformed for assembly without ATP and with DNTPs.
- picked and grew up Bca1092
To Do
- streak out JTK030 from somewhere
- transform 1934 into JTK030 and make into competent cells.
- transform 2255 into JTK030 and make into competent cells.
- EcoBam 2254 again and transform into JTK030.
- redo assembly of GFP RBS ibb.
- miniprep BCA1092 and do assembly of RFP terminator
- pick and grow up 47 to miniprep, 48
- miniprep 1934 payload.
Tahoura Samad 15:12, 28 July 2010 (EDT)
Tahoura Samad 15:12, 27 July 2010 (EDT)
IBB Work
- miniprepped RFP KA and mapped it to make sure it wasn't methylated at bgl and bam.
eco bam of rfp bam xho bgl xho. looks good
- got BCA1092 from Jin and transformed it into righty.
- miniprepped TIm's GFP.
- started assembly of AC Tim's GFP with CK rbs ibb.
- sent RBS ibb in for sequencing.
- Lifeact RFP transformations came up green, which means i used the wrong DNA. Redid Eco Bam and transformed into MG1655.
Choano Assay
- had Conor plate my transformations, but I'm worried about them actually being co transformed because top plated spec and you can see that they are all growing up around the edges, where there might not be spec. Fishy. Redid cotransformations with 1 ul and 3 ul of straight DNA of each.
Tahoura Samad 13:11, 26 July 2010 (EDT)
Choano Assay
- TetR 47 and 48 didn't grow up because I transformed them into cells without pir. They are not in PMLL, but in entry vectors. Redid those transformations today. (cotransformation)
- filtered chonaos, fed with 1 μL of overnight culture, waited 20 minutes, washed and then chased with 15 μL of white bacteria.
- observed after 1 hour, 4 hours. Still too much green bacteria outside cells, looks like they are still eating. Lots of rod-shaped. Strangely, not a lot of degradation of gfp after 1hour in cells without nh4.
- lifeact: after 1 hour, cells are green, can see rod shaped bacteria. After four hours, choanos look pretty empty (digested?) Interesting to note that several times we observed choanos with extremely round dot of green adjacent to them? Also saw balls of green in their bodies. Actin is balling up?
- did Eco/Bam of Lifeact RFP into 1256 (f4 on TIm's stock plate 1) background will be white. Will transform tomorrow.
- picked and
Tahoura Samad 13:11, 22 July 2010 (EDT)
PDD Subgroup Work
- test for competency of 3s was interesting. Cells not as good as original with 25 colonies for 2349 and about 50 for 2333. Might remake cells. Probably let them sit out for too long.
- digested rbs. ibb and gel purified product. (small fragment)
- transformations worked. Will pick 2255 on Sunday, make competent cells on Monday.
To Do
- Pick 3s, Bjh2255 on Sunday.
Tahoura Samad 12:50, 21 July 2010 (EDT)
- Lifeact assembly failed again. Found out that Jin has GFP and RFP versions of the part I'm trying to assemble. Abandoned LIfeact Assembly.
- Got Lifeact RFP and Lifeact GFP from Jin. Transformed Lifeact RFP into MC1061 and Lifeact GFP (Bjh2255)into MG1655. Lifeact RFP 2254 needs to be ecobam transfered into pbca1256.
- gel purify PCR product, digest and ligate into vector, retransform. Digestion done at 11:06 pm. Gel came up empty, so redid PCR wit 1:10 dilution of olios.
- do test transformation with 3s to make sure they work equally well as the last ones with 2333 and 2349.
- transformed Amy's new constructs into 3s. Set up choano assay for Monday.
- Get parts from Jin
- Figure out 1934 payload restructure.
- controls for competent cells looked good, with no growth on Cam, Kan and Amp and growth on LB and Spec.
To Do
- transform some of JIn's stuff into cells. Pick, grow up, miniprep. Eco Bam 2254them into 1256.
- re _eco bam transfer RFP!
- digest, ligate etc rbs.ibb and send for sequencing.
Tahoura Samad 17:03, 20 July 2010 (EDT)
- Lifeact failed to assemble.
- made competent 3 cells
- talked to Chris about choanos test with just GFP.
- Redid Lifeact assembly.
Tahoura Samad 17:03, 19 July 2010 (EDT)
- Assembly of LIfeact part
- set up FACs training.
Tahoura Samad 17:03, 16 July 2010 (EDT)
PDD Work
- did transformations of 3s with PDD
- did transformations of MG1655 with Tim's RFP (a5, plate 2). Could plate that after heatshocking because its amp.
- streaked out 3s and 5s
To Do
- Saturday: pick and grow up 3s,Mg1655 with RFP.
- Sunday: assay. Split choanos 1:5 so that we have some for monday.
- Sunday:pick and grow up RFPs so that we have samples to look at under flow cytometer.
- Monday: split choanos 1:15
Tahoura Samad 16:46, 15 July 2010 (EDT)
PDD Subgroup Work
- assembly of LifeAct abandoned. Now up to Christoph and Daniela
- learned how to use microscope at 10 am (Arkin Lab). Looked at choanos after 1 hour and got pictures
- set up time scale for weekend.
- miniprepped IBB in CK Righty and CA Righty. Will not sequence.
- go over assembly tree of that with TIm.
- got rbs oligo and resuspended at 100 mM (nm times 10 μL.)
To Do
- transformations of 3s with PDDs
- transformation of RFP into Mg1655
- start PCR of rbs onto IBB.
- split choanos so i have 50 mls on Sunday.
- streak out 3s and 5s
Tahoura Samad 13:48, 14 July 2010 (EDT)
PDD Subgroup Work
- picked and grew up 3 series for microscopy
- picked and grew up 3s and 5s for minimal media test. Same procedure as last time, but no duplicates, and grow up in minimal media with 10 mM MgSo4 overnight for 16 hours.
- Checked controls for Pir1 MC1061 lefty and rightys. Controls looked good.
IBB Work
- Miniprepped Ptet and b1006 for assembly trees
- Checked with TIm how to make oligo to put rbs on the front of ibb and made oligo. Temo of annealing region should be 55 c. THe nonsense sequence in front of the rbs is just random stuff Tim put there.
- miniprepped lifeact that is actually KA and mapped to make sure it was correct. Expected sizes 1300, 1700
To Do
- Jin wants to redo the minimal media test, but not in duplicate and make two aliquots after filtering the choanos.
- Pick 3 and 5 on Tuesday
- Split chonaos 1:15 on thursday again
- Pick and grow up CK Righty, CA Righty,RFP! and 3s.
Tahoura Samad 13:56, 13 July 2010 (EDT)
PDD Subgroup Work
- started Eco Bam transfer of 1882 part for lifeact tree into AK.
- sent Ptet+Lifeact in for sequencing. We suspect it is bad because one of the inputs was in KA, which was actually AK.
- Talked over results of minimal media testing with Jin. Results were ambiguous because there was inconstancies between the duplicates. Additionally, Jin thinks that Mg in the choano media may be obscuring the results. One thing he observed that he wants to investigate further is that he thinks 37 C might be slowing down the choano's digestion.
- Split chonaos 1:2 so they will be ready for assaying on Thursday
- Started transformations of 3s for practice assay on Thursday Morning. Talked to Jin. He said transformations are good for about 3 days on plate.
- transformed CA IBB eco bam transfer into Righty cells and CK IBB DNA (old) into righty cells.
- Checked controls for Pir1 MC1061 lefty and rightys. Controls looked good.
IBB Work
- transformed CA IBB eco bam transfer into Righty cells and CK IBB DNA (old) into righty cells.
- Made assembly trees of parts with Tim.
- picked some of Ptet and bl006 of Christoph's plates for my assembly.
To Do
- Jin wants to redo the minimal media test, but not in duplicate and make two aliquots after filtering the choanos.
- Pick 3 and 5 on Tuesday
- Split chonaos 1:15 on thursday again
- Pick and grow up CK Righty, CA Righty,RFP! and 3s.
Tahoura Samad 17:20, 12 July 2010 (EDT)
- ran 1B of colony pcr for life act again. looked good. will send for sequencing tomorrow
- ran Jin's minimal media assay
- Eco Bam transfered Ck lefty into Ca. will transform into righty cells.
Tahoura Samad 17:20, 9 July 2010 (EDT)
- miniprepped col PCR results, which grew up equally poorly. May have a hit on the first one. Will transform on Monday
- did transformations of 3s with 5 pdds.
- set up wells so amy can pick on Saturday
To Do
- Sunday: split choanos 1:5 so i have enough for Monday's testing. Set up assay for Jin, filter after 2hours. Pick and grow up 3s and 5s for 16 hrs for minimal media testing
- pick Pir1 lefty and righty for competent cell prep.
Tahoura Samad 15:18, 8 July 2010 (EDT)
PDD Subgroup Work
- direct Jin to confirm which of the combinations is actually working.
- remind Tim to email Jin about scope.
Life Act
- Even after 24 hours, the bacteria from the colony PCR still did not grow up well. Only saw growth for a couple. Miniprepped colonies 6, 9, 19, 20. Restriction mapped Bam, Eco, excpected band length of 167 and 2700
- picked MC1061 pir1 lefty and righty and am growing up in LB.
Tahoura Samad 16:10, 7 July 2010 (EDT)
PDD Subgroup Work
- nothing
- redid colony PCR with 24 new colonies. Results looked good, with many bands at expected length of 357.
- will miniprep in the afternoon.
- streaked out MC1061 pir lefty and righty since we're almost out. Will make competent cells on Friday.
To Do
- Pick and grow up Pir Lefty and Righty for competent cell prep on Friday.
- On Friday, do transformations of all 3s with all five PDDs.
- On Friday, make competent cells.
- On Saturday, take out of incubator, pick and grow up.
- On Sunday, feed to choanos.
- On Sunday, pick and grow up 3 and 5 for Monday.
- ask Tim what to do with IBB.
- get solutions of MMM and MM0 from Jin.
- ask Jin #grow up in shaker for 2 more hours. (Ask Jin what how much of each solution to grow them up in.)
- split choanos 1:5 on Sunday, so we'll have enough for Monday. Check with conor how to get 17 mL of choanos for Monday.
Tahoura Samad 18:16, 6 July 2010 (EDT)
PDD Subgroup Work/Discussion With Jin
- talked to Jin. He was not able to look at the combinations from Friday. :(
- Jin says to let all the different combinations rest, and focus on the 1934 payload.
- Jin also says to start the minimal media testing to see if pb6 works.
Minimal Media Testing
- Day 1
- Streak out 3, 5
- Pick and grow up in TB with 30 mM MgS04 (30 μL per mL) overnight.(16 hrs) Do two colonies of each 3 and 5.
- Day 2
- Dilute 100 fold by adding 30 μL of overnight culture to 3 mL of TB. (4 tubes, 2 of 3 and two of 5)
- Grow for 2-3 hours more. (2 hours)
- make 2. 1 mL aliquots of 3 and 5. (Four of each) (8 tubes total)
- Wash 3 times.
- Wash two of each with minimal media with magnesium (MMM) and Minimal media without magnesium. (MM0) Get these solutions from Jin.
- When you wash these guys, spin the cells down, carefully pipette off the supernatant without touching the pellet, resuspend with solution etc .3 times.
- grow up in shaker for 2 more hours. (Ask Jin what how much of each solution to grow them up in.)
- filter 17 mL of choanos
- incubate 8 mL of choanos with 10 μL per mL (10 mM ) NH4Cl for 10 minutes.
- feed 10 μL of bacteria to choanos. Look at after 5 hours.
- ran colony PCR for 32 colonies.
- minipreped 30 and 32, but they turned out to be contransformed. Of 32 colonies, 7 were contransformed.
- sequencing results looked okay.
- iGEM_028, CK lefty looks perfect except for a point mutation in the EcoR1 site. Looking at the abi, i think it might be a miscall.
- iGEM_029, CK Righty looks perfect except for a one base deletion after the Pts1 site. Looking at the Abi file, exactly at this point, the sample gets mixed reads. ?
- iGEM_030, CA Righty looks perfect except for the point mutation in the Ecor1 site. Looking at the abi, i think it might be a miscall.
To Do
- On Friday, do transformations of all 3s with all five PDDs.
- On Saturday, take out of incubator, pick and grow up.
- On Sunday, feed to choanos.
- On Sunday, pick and grow up 3 and 5 for Monday.
Tahoura Samad 13:22, 5 July 2010 (EDT)
- picked and grew up 1, 2, 6, 7, to make competent cells.
Tahoura Samad 13:22, 2 July 2010 (EDT)
- most of my transformations with the new competent cells were successful.
|Competent cell
|Growth in initial competent cells
|Growth in Small Scale Competent Cells
|Result of Assay with Jin
|tons of colonies
|transform new, assay
|tons of colonies
|transform new, assay
|1 colony (twice)
|tons of colonies
|transform new, assay
|50-100 colonies
|transform new, assay
|100-200 colonies
|transform new, assay
|pBca156-Bjh2343 with BAC
|15 colonies
|pBca156-Bjh2343 with BAC
|2 colonies
|pBca156-Bjh2343 with BAC
|4 colonies
|pBca156-Bjh2343 with BAC
|pBca156-Bjh2343 with BAC
|tons of colonies
|make new, transform, assay
|tons of colonies
|make new, transform, assay
|12 colonies
|make new, transform new and old, assay
|6 colonies
|make new, transform new and old, assay
|7 colonies
|make new, transform new and old, assay
|pBca156-Bjh2344 with BAC
|tons of colonies
|pBca156-Bjh2344 with BAC
|tons of colonies
|pBca156-Bjh2344 with BAC
|1 colony (twice)
|100-200 colonies
|pBca156-Bjh2344 with BAC
|1 colony(twice)
|35-50 colonies
|pBca156-Bjh2344 with BAC
|35-50 colonies
|100-200 colonies
|transform new, assay
|35-50 colonies
|<100 colonies
|tons of colonies
|transform old, assay
|tons of colonies
|transform old, assay
|tons of colonies
|transform old,assay
|pBca156-Bjh1934 with BAC
|tons of colonies
|pBca156-Bjh1934 with BAC
|tons of colonies
|pBca156-Bjh1934 with BAC
|tons of colones
|pBca156-Bjh1934 with BAC
|1o colonies
|pBca156-Bjh1934 with BAC
|tons of colonies
|transform old, assay
- competent cell checks for new 1 and new 6 looked good.
- miniprepped IBB as CA lefty, CK lefty and CK righty, and sent out for sequencing with ca998.
Tahoura Samad 13:38, 1 July 2010 (EDT)
- controls for JTK030 looked good with no growth on Kan, Amp, Cam, good growth on LB, and a little growth on Spec, which is the normal result because of mutations in the bacteria. Those are labeled with double black stripes, in a box in the back of the -80 in the 2nd row from the top.
- picked and grew up the results of PCA for the new nuclear localization tag. They had plenty of colonies, with only one background green colony. Will miniprep them tomorrow.
- did small scale competent cell prep of 1 and 6.
- did the following transformations (1old:2348), (5 old:2291) (6 old:2291) (6 new:2291),(3 old :2348) (3 old: 2349) (3 old:2291) (2 old:2348) (2 old: 2349)(2 old:2291) (2 new:2348) (2 new:2349) (2 new: 2291), (1 new:2331) (1 new:2333) (1 new:2348) (1 new:2349) (1 new: 2291) (6 new 2348) (6 new:2349), (IBB into lefty).
- redid assembly of stage 0 of life act but with 2.4 μL of each DNA and 1.2 μL of 5x NEB2.


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text:
VerHagen becoming viable starting pitcher
No. 15 prospect in system has to work on curveball, changeup to be more effective
Not unlike every other prospect, Drew VerHagen is a work in progress. It takes time to develop a complete pitch repertoire and refined command and control.
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text:
ASU Electronic Theses and Dissertations
This collection includes most of the ASU Theses and Dissertations from 2011 to present. ASU Theses and Dissertations are available in downloadable PDF format; however, a small percentage of items are under embargo. Information about the dissertations/theses includes degree information, committee members, an abstract, supporting data or media.
In addition to the electronic theses found in the ASU Digital Repository, ASU Theses and Dissertations can be found in the ASU Library Catalog.
Dissertations and Theses granted by Arizona State University are archived and made available through a joint effort of the ASU Graduate College and the ASU Libraries. For more information or questions about this collection contact or visit the Digital Repository ETD Library Guide or contact the ASU Graduate College at firstname.lastname@example.com.
- Arizona State University
- Nelson, Brian C
- 1 Hsiao, Ihan
- 1 Kobayashi, Yoshihiro
- 1 Kury, Nizar
- Virtual
- Learning
- 1 Adaptive
- 1 Computer science
- 1 Education
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- 1 Game
- Reverse Fountain Cytoplasmic Streaming in Rhizopus Oryzae
- Diné Research Practices and Protocols: An Intersectional Paradigm Incorporating Indigenous Feminism, Critical Indigenous Research Methodologies and Diné Knowledge Systems
- Developing an Augmented Reality Solution for Mapping Underground Infrastructure
- Characterization of 2D Human Ankle Stiffness during Postural Balance and Walking for Robot-aided Ankle Rehabilitation
- Using Molecular, Cellular and Bioengineering Approaches Towards Understanding Muscle Stem Cell Biology
Programming is quickly becoming as ubiquitous and essential a skill as general mathematics. However, many elementary and high school students are still not aware of what the computer science field entails. To make matters worse, students who are introduced to computer science are frequently being fed only part of what it is about rather than its entire construction. Consequently, they feel out of their depth when they approach college. Research has discovered that by teaching computer science and programming through a problem-driven approach and focusing on a combination of syntax and computational thinking, students can be prepared when entering higher …
- Contributors
- Kury, Nizar, Nelson, Brian C, Hsiao, Ihan, et al.
- Created Date
- 2017


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With their new Windboiler, MSR has definitely done a great job on the "human factors" (better handle, better lid, a cup that's actually useful, more slots so the pot attaches more easily, etc.), but if the Windboiler isn't significantly better than a Jetboil at handling wind, then it's really just a prettied up Jetboil.
So, how well does a Windboiler handle wind? I decided to find out.
I did several head to head tests with a Jetboil Sol in light to moderate winds. I could see that the Windboiler remained fairly constant while the Jetboil would lose ground (boil time would lengthen) as the winds increased, but I didn't really have the sense that I fully understood the capabilities of the Windboiler. Time to put it to a real test I thought.
So I headed to the windiest spot I could think of in Southern California – the San Gorgonio Pass area near Banning, CA, home to many a wind turbine.
I picked a spot for testing at the mouth of a canyon where I thought winds would come whistling through. I was not disappointed.
I have put up two videos on my blog that I think show the difference in the way a Jetboil behaves vs. a Windboiler in significant winds. Have a look if you like.
MSR Windboiler – Wind Testing


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« Two Great New Reports Look at Teacher Effectiveness | Main | New Data Show Why We Need to Start Early to Keep Kids From Falling Behind »
Texas Pre-K Programs Improve Kids' Elementary Achievement
Really important new CALDER study finds evidence that participation in Texas' state-funded pre-k program is associated with increased 3rd grade math and reading scores for participating youngsters, as well as reduced rates of grade retention and special education placement.
This is a very important finding for several reasons: First, Texas enrolls more kids in pre-k--224,000--than any other state in the country, so this evidence indicates that a lot of kids are being helped.
Second, this study should definitively put to bed the (mis)conception among a lot of K-12 education reform types that the evidence on pre-k effectiveness is shaky or based only on small, boutique programs. Yes, a decade ago the argument for pre-k was based primarily on evidence from the small samples in the High Scope/Perry Preschool and Abecedarian programs. But that hasn't been true for a while now. Today we have evidence of strong positive impacts from the Chicago Child Parent Centers, New Jersey's Abbott Pre-K Program, and Oklahoma's UPK program--all large scale programs funded with relatively modest amounts of public funds. And we have similar positive evidence on a growing number of state-funded pre-k programs. With over 200,000 kids participating and an average per pupil spending of $3,761 per participating child, no one can say that Texas's pre-k program is a small, boutique program that can't be widely replicated. Texas is the definition of scale, and the level of resources in play here is modest.
Third, this study is particularly powerful because it finds evidence of impact on test scores in 3rd grade--four years after children participated in the pre-k intervention. Thus, Texas joins New Jersey as one of two (very different) states where we have evidence that pre-k participation improves academic achievement into elementary school. That's important, particularly because "fade-out" of test score gains is commonly cited by opponents as a reason not to fund pre-k. Yes, some studies find evidence that pre-k or Head Start learning gains fade out, but the overall body of evidence is much more complex and suggests that some benefits do last into the elementary grades and beyond. We need to move from using "fade out" as a rhetorical tool in policy conversations to focus on identifying the policies and practices--in both pre-k and the early elementary grades--that maximize the extent to which pre-k gains are sustained through elementary school. And we need to fund a lot more kids to attend pre-k in the first place.
Fourth, this study is significant because it's the first to look at the impact of pre-k on children tested in both English and Spanish--and finds positive results of pre-k participation for Spanish-speaking youngsters who take the Texas state tests in Spanish because their English is not good enough to take them in English. As the authors note, this finding is particularly critical given the demographic changes currently taking place in our country and the growing numbers of children born in families where English is not the home language.
Finally: While not part of the study finding per se, I hope this study will encourage people to think seriously about how we define and talk about "quality" in pre-k. The authors note that Texas' pre-k program is not a "high-quality" pre-k program, in that it involves relatively low per-pupil resources and scores poorly on the National Institute for Early Education Research's report card, which focuses primarily on indicators of pre-k resources and inputs. But in another sense, the Texas Pre-k program incorporates a strong focus on instructional strategies and content targeted to school readiness, and many Texas Pre-k classrooms have benefited from Texas School Ready, an intervention that equips programs with a research-based curriculum focused on content that predicts school readiness, provides extensive professional development and coaching to pre-k teachers, and uses performance monitoring to track students' progress towards school readiness and inform instruction. All of the evidence I've seen suggests that the extent to which pre-k programs focus on content that predicts school readiness, support teachers in understanding and using effective instructional techniques for young children, and utilize developmentally appropriate measures to inform instruction are just as if not more important than the input-driven quality indicators (bachelor's degrees, class sizes) that pre-k policies typically focus on. Yet our policy debate and state and federal policies around early childhood tend to focus heavily on the latter set of indicators rather than the former. A smart policy discussion needs to focus much more on how to create the conditions for effective, developmentally appropriate instruction in pre-k classrooms.
All that said, this study is an important addition to the body of evidence that makes pre-k the education intervention with the strongest body of evidence of effectiveness, and one that we as a nation are idiots not to have spent a lot more money on over the past decade when spending on K-12 schools was skyrocketing with little to show for it.


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Christmas Customer Service Information
Our running shoes have a variety of support, ranging from neutral to stability. We're passionate about meeting your needs and ensuring the best running experience. Whether you're looking for road, trail, fell, hill, cross-country or track running shoes, we have a large selection to suit your needs. Our trainers are priced to accommodate different budgets so you'll always be able to find an affordable pair.
14 Items


text: Penicillin-Streptomycin Solution (ATCC® 30-2300™) General Information Quality Control Specifications Documentation Print Email Share Share this product Facebook Twitter Google+ Product Format frozen 100 mL Storage Conditions -20°C or colder Applications 10,000 I.U./mL Penicillin, 10,000 (μg/mL) Streptomycin For reducing the chances of microbial contamination in cell culture. Between 0.5 and 1 mL of Penicillin-Streptomycin solution are added to 100 mL of cell culture media for a final concentration of 50 to 100 I.U./mL penicillin and 50 to 100 (μg/mL) streptomycin. Comments NOTE: Most cell culture technologists avoid using antibiotics and antimycotics for routine culture work. Antibiotics and antimycotics may mask contamination by susceptible bacteria and fungi while permitting mycoplasma to flourish unnoticed. Antibiotics and antimycotics may interfere with the metabolism of sensitive cells in culture. However, one may elect to introduce antibiotics and antimycotics for short periods to primary cultures or as a safeguard while propagating specific valuable stocks. Biosafety Level 1 Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country. Quality Control Specifications Lot-specific test results are listed in the Certificate of Analysis, which is available at www.atcc.org or by contacting ATCC Technical Service. SPECIFICATION CERTIFICATE Test Specification pH1 Report results Osmolality2 Report results Endotoxin3 ≤ 0.25 EU/mL @ 1:100 Antimicrobial Activity4 (1:100) Inhibition of S. aureus & E. coli Sterility5 Pass Measured with a standard pH meter. Meters are calibrated daily using standards traceable to the National Institute of Standards Technology using guidelines outlined in the current edition of USP. Measured with a standardized osmometer, i.e. freezing point depression. Meters are calibrated daily using standards traceable to the National Institute of Standards Technology using guidelines outlined in the current edition of USP. Chromogenic LAL assay using guidelines outlined by the manufacturer & FDA (December 1987), "Validation of the Limulus Amebocyte Lysate Test as an End Product Endotoxin Test for Human and Animal Parenteral Drugs, Biological Products, and Medical Devices". Measured using Minimal Inhibitory Concentration (MIC). At 100 CFU/mL, MIC must meet user concentration. Tested by a modified membrane filtration technique. Basic Documentation Product Sheet Certificate of Analysis SDS


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Exclusive Message for Fantasy-Faction users from Scott Lynch
So, we've heard the news that Scott Lynch's latest novel is out this year (whoo!), but now we've managed to track Scott down, drag him to a keyboard and force him to type something for us. We've edited out the cries for help and left you with the awesome-ness that is the kind words of Scott Lynch.
But before we begin, the CONFIRMED publication date of the book in the UK is Thursday, October 10th, 2013 and around the same time in the USA too!
Please do take the time to comment and thank Scott for stopping by. You never know, we might convince him to do it again. 😉 Here he is:
– – –
Hello, Fantasy-Faction readers!
My publishers and I have been sitting on some news for about a week and a half now, and I've been ready to spontaneously combust from keeping quiet. Which is, I suppose, a small cosmic justice for the fact that everyone else has had to wait five and a half years to find out what happens next for Locke Lamora.
Well, as you probably know by now, what happens next is called THE REPUBLIC OF THIEVES, and it hits shelves in the UK on October 10th.
I have been open elsewhere about the nature of the delays in the writing of this book; it's been a difficult and occasionally ugly span of time, and while everything has brightened considerably in the last year or so, it's hard not to feel that depression and anxiety have stolen a chunk of my life outright, a chunk I would have preferred to use giving you more books. But it is what it is, and I like to think that there is some compensation: The version of THE REPUBLIC OF THIEVES you'll get is a tighter, more carefully considered, and (or so I flatter myself) more mature version than anything I might have handed in before.
Or so my cat tells me, and the voices in my head agree. And if you can't trust the voices in an author's head, who can you trust?
I know that readers have a sensibly-honed caution about long series that languish or threaten to go unfinished. I hope to get mine back on course with a vengeance, lest it become another task in Brandon Sanderson's inbox. The poor guy is busy enough.
So, THE LAST DANGEROUS V… Er, I mean, THE REPUBLIC OF THIEVES! October 10th. And now it's some other writer's turn to wear the Crown of Lateness. I have already started work on the fourth Gentleman Bastard book, THE THORN OF EMBERLAIN.
Cheers! I'll be in the UK this year for the World Fantasy Convention, and hopefully touring as well. I hope to see more than a few of you!
– – –
I'd like to say WELCOME BACK TO THE GENRE, Scott. We missed you mate and I hope you're feeling better in yourself. Everyone here at Fantasy-Faction is behind you (staff and visitors) and grateful for the wonderful world you've given us. You can be sure that we'll be the first in the queue at bookshops in October and first hunting you down to receive handshakes, hugs and high-fives when you land in the UK!
Coming October 2013:
43 Comments
Brilliant news Scott! So glad you are back! Wahoo!
Great Scott!
That's fab news, thanks & best wishes. Look forward to shaking your hand in the UK some day soon.
Now to go re-read the first two….
" And now it's some other writer's turn to wear the Crown of Lateness."
Can I borrow that crown please, I think I'm gonna need it.
I have one thing to say… YAY!
Thanks for the message Scott. You have been sorely missed, but as intelligent genre readers, we understand that good things come to those who wait (Sanderson doesn't count. I've no idea how he can write as much as he does while maintaining quality). Looking forward to RoT!
I guess I can stop lying face down on the floor, softly moaning because of extreme Jean deprivation.
On second thought, I think I can keep that up until October. Cheers, Mr. Lynch. This was awesome news to wake up to. Congratulations!
* extreme Jean deprivation. Me too. I feel for you – roll on October!
Good to see you back mister! So glad to hear you're in a better place, been rooting for you. Will definitely make the effort to come see you in the UK 🙂
That gone straight on to my To Read list. Excellent news
Amazing! Can't wait to meet you at WorldCon, Scott
I for one would like to welcome the return of our Scott Lynchian Overlord *bows in homage*
Oh that's brilliant news! So glad things are feeling brighter for you too 🙂 *coughcometoBristolConcough*
So glad to have you back, Scott! Brilliant news.
I'm so overjoyed I don't know what to do with myself! 😀 I've tried to write this four times now and I still can't put to words how happy I am that Scott is feeling better and back in business and that I have something fantastic to look forward to in October!
But yes, that's what it is, in a nutshell. I'm now trying to round up some fellow fans and seeing if I can drag them out for drinks to celebrate!
Can I just say (as Scott's UK publisher) how grateful I am to all of you for your continued enthusiasm for Scott's books but, more importantly, the kindness and care that you've shown him in the years since Red Seas and now, in response to the announcement and to Scott's message. I know how much it means to him. Thank you!
Well done, Scott! You helped inspire me to write my own novel. I'll always remember sitting on the beach in Argentina in 2009 utterly transfixed by the adventures of Locke and co.
[…] […]
You're the man, Scott!
And yep, better write your own books – you don't want to be responsible for the Stormlight Archive volumes coming out later. 😉
Thanks Scott. It's great to hear that you are getting over the worst of your health problems. I've had issues with depression myself, so I know how tough it can be.
I loved the Lies of Locke Lamora, but did not want to read the sequel while the series was up in the air. I didn't like the idea of reading Red Seas Under Red Skies and then being out of Locke Lamora books. As soon as I heard this publication news I snapped up that book and look forward to reading it soon.
Thank you Scott, for all your great work. You, sir, are an inspiration for us all!
Thanks Scott! Also, that bit about Brandon Sanderson's inbox slays me.
Too busy jumping up and down excitedly to type much – very, very happy news! Must carefully schedule reread of Lies of Locke Lamora and Red Seas Under Red Skies to segue straight into Republic of Thieves when it's published. Beautiful cover too!
Love you Scott! Come to Sweden too
Great news!
I can safely say that without the Locke Lamora books, I wouldn't have got back into fantasy in such a big way. Thank you and welcome back, Scott.
Wow this is an amazing 17th birthday present, great news! 😀 You're a brilliant writer Scott and, although I've not met you personally (I'm hoping to when you come to the UK), you seem like an amazing person too. You also have really cool hair.
Thanks again Scott, peace.
Seriously, best news ever! Love the Sanderson comment, too.
Same here.
I'm so happy you're back on track Scott, this news made my day!
P.S. Nice comment about Brandon there. 😛
[…] in the last post, and I think I'll mention it again, because I could not be more thrilled: Scott's handed in the final revisions on The Republic of Thieves, and its publication date is set for October 10th 2013 in the UK and October 8th 2013 in the […]
Fantastic News! I've bought the first two 3 different times now, (I keep giving them away) and knowing there's new stuff on the way means I'll have to buy them all over again!
Thanks Scott really looking forward to another Locke Lamora book
This has made my year. 🙂
Time for a reread later on in the year I think.
This can be summed up as: SWEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEET
So grateful that I fell upon Scott's work a few years back, the wait hasn't been so long for me – but REALLY excited. Thank you Scott 🙂
Congratulations, Scott! While I don't wish that kind of delay on anyone, it is comforting for people like me to know sometimes books take time, lots of time (3.5 years on my novel). I hope things are on the up for you.
I for one know exactly what it's like to feel like depression and anxiety have stolen a part of one's life, so I can only say that I'm proud of you for overcoming it! That's no easy feat and both me and my fiancé (who isn't a big reader but adores your books) are eagerly awaiting more. 😀
Hurray! My man and I are terribly excited, having truly enjoyed your first 2 books. With that said, your work is worthy and if this happens to be all we get, we will enjoy it, savor it, for many years to come. And I personally don't mind waiting for a the author to say, 'OK, this is good to go.' Take as much time as you need. I will happily be waiting in a corner booth with some hot tea and a well-worn copy of The Lies of Locke Lamora.
Hey, all– I just wanted to echo what Simon said… thank you for all the kind thoughts and good wishes. They've been humbling and edifying. And if you end up enjoying the book when it arrives, spare a kind thought for Simon, as well… he's endured a great deal behind the scenes to deliver it to you. 🙂
Twitter: @scottlynch78
[…] six years, lost to depression and anxiety attacks. (He talks about it here, on Fantasy Faction – http://fantasy-faction.com/2013/exclusive-message-for-fantasy-faction-users-from-scott-lynch Luckily he is feeling more upbeat these days, but that kind of illness, or indeed any serious […]
This is wonderful, wonderful news. Fantastic that the next book is on its way, can't wait for that, but even better that you have overcome the depression and found your strength. Wishing that you go from strength to strength now, believe me you will look back on those days as building blocks, despite the feeling now that they stole away your time.
Looking forward to seeing you in Brighton!
[…] Exclusive Message from Scott Lynch to Fantasy-Faction – A message from Scott announcing RoT […]


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O's agree to terms with Cuban prospect Alvarez
Cuban outfield prospect Dariel Alvarez has agreed in principle to a deal with the Orioles worth $800,000, he told MLB.com on Thursday.
Alvarez, who is waiting on the results of his physical, could officially sign on Saturday. He met with Orioles officials earlier this week at the club's Spring Training complex in Sarasota, Fla.
"I'm obviously very happy to get my career going," Alvarez, 24, said from Miami. "Now, it's time to get to work."
Alvarez, a right-handed hitter who played for Camaguey in Serie Nacional in Cuba, defected from the island last summer and later played professionally in Vera Cruz, Mexico. He was declared a free agent by the Commissioner's Office in January, and was cleared by the U.S. government to enter into a contract with a big league club the next month. Alvarez said a change in representation is part of the reason his appearance on the market was delayed. Alvarez admitted that he considered returning to Cuba, but changed his mind.
"It's been very hard with a year full of ups and downs, but I knew it was best if I stayed here," said Alvarez, who starred in a showcase in front of scouts last month in Florida. "I give thanks to God that it all worked out. I'm ready. I'll go to whatever level they want me to go."
Because Alvarez is at least 23 years old and has played at least three seasons in a Cuban professional league, he is not subject to the new international signing guidelines established by the Collective Bargaining Agreement.


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Sorry if the question sounds flippant, it isn't meant to be, and I know that this is asking an opinion rather than a verifiable fact but bear with me...
I'm looking for somewhere to start uploading some of my better older photos and some of my new photos, and also to group photo project photos together and that kind of thing.
I know how Flickr works, I don't think it's the best of the photo-sharing sites I've seen, but it's ok and the crucial thing is I want to use to follow/share my pics with other photographers and in high resolution (I already have Facebook for sharing photos with friends). Flickr seems to be the best place for this as it's so well established with loads of users.
However I've heard a few people mention that 'flicker is dying' and that photographers are starting to exodus to other services. I don't want to commit to a service that is in decline, and although nobody can predict the future, can anybody give me an opinion on this?
(additionally, although I'm not looking for a review of Flickr, if anybody knows of any extremely important reason to either use it or not use it that I may have overlooked, please let me know) (I do know about the upload limits for free accounts)


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I have a subdirectory within my WP installation called
labs. I created a WP page called
labs as well. When a user hits
mydomain.com/labs/ I want them to load up the WP page. But instead it's loading up the
labs directory listing.
I've read a few ways to do this if I want my WP page to have a different name than my directory name, but I don't really want that. For example, I want to have:
mydomain.com/ <-- WP Root
mydomain.com/labs/ <-- Pretty WP page listing all of my projects
mydomain.com/labs/project1/ <-- raw (i.e. no WP) web app
mydomain.com/labs/project2/ <-- raw (i.e. no WP) web app
Is this possible?
I should also mention that WP is installed in the root of
mydomain.com, so
/labs/ is both a WP page and a subdirectory that I created manually.
I renamed my
labs folder to
projects and used the following .htaccess.
RewriteEngine On
RewriteCond %{REQUEST_URI} labs/(.+)$
RewriteCond %{REQUEST_URI} !labs/(.+)/$
RewriteRule ^labs/(.+)$ http://mydomain.com/labs/$1/ [L]
RewriteRule ^labs/(.+)$ projects/$1 [L]
I'm a little unclear as to why I had to use
RewriteRule ^labs/(.+)$ http://mydomain.com/labs/$1/ [L]
instead of just
RewriteRule ^labs/(.+)$ labs/$1/ [L]
But when I used the latter it never appended the trailing slash.


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24 new species of flower fly found in Central and Southern America
A team of scientists have described twenty four new species of dipterans belonging to Quichuana genus, of which only a further 24 species were known. The researchers, including two Spanish biologists, have been studying the forests of Central and Southern America for ten years and they have now published their results in the Zoological Journal of the Linnean Society.
A ten-year study in forests of the American continent has resulted in the description of 24 new insect species from the Quichuana genus that are also known as 'flower flies'.
Up until now only a further 24 species were known and this genus belongs to the Syrphidae family, which is a group with similar characteristics to that of bees and wasps but with a different taxonomic order. The study has been published in the 'Zoological Journal of the Linnean Society'.
As explained to SINC by María Ángeles Marcos-García, researcher at the Ibero-American Biodiversity Centre (CIBIO) of the University of Alicante and one of the authors of the study, the species of the Quichuana genus are "not well known as they live in tropical forest areas where insect studies are scarce."
Numerous studies in these forests have been carried out in the last ten years that aim to "understand and provide useful data for supporting conservation measures in those areas that are home to such high levels of biodiversity," explains the biologist.
Double the amount of Quichuana species
Thanks to these studies, 24 more species have been discovered, meaning that the number of documented species in this genus has doubled.
"Some specimens were captured as adults while in flight whereas others were taken as larva and were then raised in our laboratories as adults," outlines the researcher.
In this way it was possible to find out how larvae "live in small cumuli of water retained in different parts of the plant" and how "a relationship has been established between the insect species and their plant development environment," adds the researcher.
According to the author the discovery of this insect-plant relationship is "especially important for establishing conservation methods, both for these species and others that share the same development environment."
According to the entomologist, "hoverflies are present in practically all terrestrial ecosystems and they carry out very important biological functions, such as pollination, nutrient recycling and biological control of plagues as their larvae feed on other insects that are damaging to crops and ornamental plants, such as plant louses."
Explore further


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How to sell images
YAY Image Agency connects photographers with potential customers. At YAY photographers upload their images, and we present them to potential clients for licensing. When your image is licensed to a customer who buys credits, you get a 50% commission. We are looking for all kind of images - the most popular are lifestyle, people, objects, food and travel.
Learn more:
Make Money on Our Referral Program
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We at YAY are always looking for great photographers and illustrators!
Professional, semi-professional and amateur photographers are all more than welcomed at our site. We are looking for a wide variety of images and illustrations, both commercial, editorial and art.
On this page you'll find information on how you registrer at YAY, how you upload and sell your images, what commission you'll receive and how you can promote your images. You'll find more general information about YAY at our About Us page, and answers to general questions in our Image FAQ. If you can't find an answer to your questions, please contact us at firstname.lastname@example.com.
We are continuously updating our site, and we are planning to release several features for photographer this year. If you have any requests or ideas we'd love to hear from you, e-mail us at firstname.lastname@example.com. You can follow us on Facebook to get a day-by-day update.
We hope you'll join YAY, and that you'll enjoy being a part of our community!
The commission is 50 % to contributors on all single image sales. For subscriptions the commission is 1,5 euro per download for high-res, €0.75 for medium sized images and €0.20 for small images.
On all our partner sales we offer you a 50% commission. Our partners license your images at different prices, ranging from microstock prices like YAY's, and up to several hundred dollars for an image. You will always get a 50% cut on what we get for a license!
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We use PayPal for payouts, if PayPal is not available in your country – please contact us on email@example.com and we'll arrange another payout option for you (we also support MoneyBookers). Payout limit is 30 euro.
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1. Sign Up First, you have to sign up, and then you are ready to begin uploading.
2. Upload Your Files Click on My Account, and then choose Upload. Now you can upload from within the browser (press "Click here to upload files) or with FTP (server: ftp.yaymicro.com). If you have a vector-file you can upload a zip with the jpg to get the IPTC-data.
3. Describe Your Images. After your files have been uploaded, click "Describe".
4. Add Releases You only have to do this once for each model! If you have images of people you need a signed contract allowing you to license the photo. You can use your own, forms from other agencies, or ours: Model Release.doc or Model Release.pdf
To add a model or property release click on "Releases" in the left menu. Click "Add model release" or "Add property release". Fill out the fields:
Remember that these fields will be used in searches, except name and optional fields.
The model release can now be added to all of the images of this model. You only need one model release for each model.
6: After Submitting
Images you have submitted that are waiting for review will be listed here.
If an image has been rejected it will show here. See our list of rejections to find more information. If you can correct the error you are welcome to resubmit the image.
Approved images that are ready for sale will be listed here. You will be able to change title/description/keywords/country here. If you need to you can also delete an image.
Send Files by Mail
If you have a large amount of material you can send your material to us on a CD, DVD or portable hard drive:
YAY Media AS
Gaustadalléen 21
Image Requirements
Images need to be at least 6 megapixels and in addition a minimum of 1500 pixels on each side. For example: 4000x1500px, 1500x4000px or 3000x2000px.
We accept JPEG, JPEG 2000, SVG and EPS (should be AI8 compatible .eps file).
Please note: You do not have to upload a JPEG-file with your vector. Our system automatically creates a JPEG. If you wish to use a JPEG for IPTC data you can zip a jpeg and eps (or svg) together and upload that and any IPTC information found in the jpeg will be put on the entries on the describe page.
Make Money on Our Affiliate and Referral Program
Read more at our Affiliate Program Page. We offer 20 % commission on all customer referrals, €0.1 on all photographer referral sales, and more!


text:
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14-days Return Policy. The item must be undamaged, unworn, and unused with tags still attached within 14 days of the notification that the item will be returned. We will provide a return/refund policy for any damaged or defective items as long as any claims are placed within the agreed-upon 5 days of the item being received.
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A few days ago, Kotaku posted a gallery of concept art from a rejected pitch for a steampunk Batman videogame based on the Elseworlds comic Gotham by Gaslight. So what exactly did we miss out on?
The Elseworlds comics are DC's alternate universe books, "What if?" scenarios featuring familiar superheroes. Gotham by Gaslight was the first Elseworlds book, and planted Batman in the Victorian era. The original Gotham by Gaslight story was written by Brian Augustyn, with pencils by Hellboy creator Mike Mignola.
Artist Julie Farrell posted concept artwork from a Gotham by Gaslight game she was working on with Day 1 Studios (although you'll have to go to Kotaku to see the images, since Farrell was asked to take them down). Farrell's designs look pretty heavy on the steampunk, with plenty of brass and gears.
I can't help but wonder how closely a Gotham by Gaslight game really would have followed the original story, which is a tightly written detective tale featuring Jack the Ripper, who has fled Whitechapel for Gotham City. (His timing really couldn't have been worse, since that's precisely when Bruce Wayne starts donning his cloak and cowl.) Augustyn and Mignola's Batman is also pretty light on the gadgets, a far cry from the clockwork batarang. Perhaps Farrell and Day 1 drew their inspiration from the Gotham by Gaslight follow-up, Master of the Future, which featured a supervillain with an airship and an automaton sidekick.
Or maybe the Gotham by Gaslight videogame would have simply been a generically steampunk version of Batman that borrowed the Elseworld comic's name and featured steampunked versions of Batman's rogue's gallery. In that case, I still hope they would have borrowed some of Mignola's visual ideas — the leather-outfitted Batman with high-necked coat over his costume, and perhaps his strychnine-poisoned Joker with the rictus grin.
Get a Glimpse at an Elseworlds Batman in the Cancelled Gotham by Gaslight Game [Kotaku]


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]]>Bran Muffins]]>
]]>Garlicky Lentil Soup]]>
]]>Banana-Strawberry Smoothie]]>
Vegetable-oil cooking spray
1 cup All-Bran fiber cereal
1 cup raisin bran cereal
1/4 cup skim milk
1 1/4 cups fresh orange juice
1 1/4 cups all-purpose flour
1 tablespoon baking powder
1/4 teaspoon cinnamon, or to taste
1/4 teaspoon salt
1 large egg white, beaten lightly
1/2 cup honey
Preheat oven to 400°F and lightly coat nine 1/4-cup muffin tins with cooking spray.
In a large bowl combine cereals, milk, and orange juice and let stand 5 minutes. In a small bowl whisk together flour, baking powder, cinnamon, and salt. Into cereal mixture stir egg white and honey until combined well. Add flour mixture and stir until just combined. Divide batter among muffin tins and bake in middle of oven 20 minutes, or until golden and a tester comes out clean. Turn muffins out onto racks and cool.
Makes 9 muffins
Source: Gourmet, Epicurious.com
8 large garlic cloves
4 cups cold water
2/3 cup lentils (preferably French green lentils)
1 tablespoon chopped fresh thyme leaves
2 tablespoons chopped fresh flat-leafed parsley leaves
1 1/2 to 2 teaspoons salt
extra-virgin olive oil to taste
red-wine vinegar to taste
Slice garlic. In a 2-quart saucepan bring water to a boil with garlic, lentils, and thyme and simmer until lentils are tender, about 25 minutes. In a blender coarsely purée mixture (use caution when blending hot liquids) and return to pan. Stir in parsley, salt, and pepper to taste and bring soup to a simmer.
Serve soup drizzled with oil and vinegar.
Makes about 3 cups, serves 2
Source: Gourmet, Epicurious.com
Last reviewed September 2005 by Steven Bratman, MD]]>
Please be aware that this information is provided to supplement the care provided by your physician. It is neither intended nor implied to be a substitute for professional medical advice. CALL YOUR HEALTHCARE PROVIDER IMMEDIATELY IF YOU THINK YOU MAY HAVE A MEDICAL EMERGENCY. Always seek the advice of your physician or other qualified health provider prior to starting any new treatment or with any questions you may have regarding a medical condition.
Copyright © 2007 EBSCO Publishing All rights reserved.


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Have you ever felt that you want to escape reality? Bobby is a sixteen year old single parent who sometimes wants to be set free. He loves his daughter so much, but he is struggling to deal with the tough battle of having all the burden of raising a little girl on his shoulders.
The First Part Last by Angela Johnson alternates between Bobby's life pre-baby and post-baby. As it progresses you understand the sights, sounds, and images that constantly make him think about how his life has changed since he became a father. At the same time he loves his daughter and is determined to take care of her. What will Bobby do? Will he lose himself? Where is the baby's mother? Intrigued? The First Part Last is different than most teen pregnancy stories because it is told from the father's point of view and it will definitely make an impression on you!


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Im working on a website where I need to create a submenu that shows only the parent pages.
When I click on one of the parents it has to show the children from that parent.
Here is the menu hierarchy I used this widget plugin: Subpages Extended for showing the parents and children:
- Parent
- Parent1 from parent
- Child1 from parent1
- Child2 from parent1
- Parent2 from parent
- Child1 from parent2
- Child2 from parent2
Could you explain me or give an example/tutorial on how to do it?