- Mar 2021
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www.cell.com www.cell.com
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Most Suspected Brugada Syndrome Variants Had (Partial) Loss of Function
AssayResult: 3.4
AssayResultAssertion: Abnormal
ReplicateCount: 22
StandardErrorMean: 0.8
Comment: This variant had loss of function of peak current (<10% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)
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we selected 73 previously unstudied variants: 63 suspected Brugada syndrome variants and 10 suspected benign variants
HGVS: NM_198056.2:c.1106T>A p.(Met369Lys)
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- Feb 2021
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www.ncbi.nlm.nih.gov www.ncbi.nlm.nih.gov
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RRID:ZDB-ALT-170927-7
DOI: 10.7554/eLife.54491
Resource: (ZFIN Cat# ZDB-ALT-170927-7,RRID:ZFIN_ZDB-ALT-170927-7)
Curator: @scibot
SciCrunch record: RRID:ZFIN_ZDB-ALT-170927-7
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RRID:ZDB-ALT-110215-7
DOI: 10.7554/eLife.54491
Resource: (ZFIN Cat# ZDB-ALT-110215-7,RRID:ZFIN_ZDB-ALT-110215-7)
Curator: @scibot
SciCrunch record: RRID:ZFIN_ZDB-ALT-110215-7
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elifesciences.org elifesciences.org
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RRID:ZFIN_ZDB-ALT-120221-7
DOI: 10.7554/eLife.53403
Resource: (ZFIN Cat# ZDB-ALT-120221-7,RRID:ZFIN_ZDB-ALT-120221-7)
Curator: @scibot
SciCrunch record: RRID:ZFIN_ZDB-ALT-120221-7
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Annotators
URL
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jmg.bmj.com jmg.bmj.com
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Supplemental material
AssayResult: 101, 106
AssayResultAssertion: Normal
Comment: See Table S3 for details
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Supplemental material
AssayResult: 4, 5
AssayResultAssertion: Abnormal
Comment: See Table S3 for details
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We analysed a total of 82 blood samples derived from 77 individuals (online supplemental table 3). These 77 individuals corresponded either to new index cases suspected to harbour a pathogenic TP53 variant or to relatives of index cases harbouring TP53 variants.
HGVS: NM_000546.5:c.1054G>T p.(Asp352Tyr)
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onlinelibrary.wiley.com onlinelibrary.wiley.com
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RRID:ZFIN_ZDB-GENO-190822-7
DOI: 10.1002/dneu.22791
Resource: (ZFIN Cat# ZDB-GENO-190822-7,RRID:ZFIN_ZDB-GENO-190822-7)
Curator: @Naa003
SciCrunch record: RRID:ZFIN_ZDB-GENO-190822-7
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10.11.66.200 10.11.66.200
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Full Card Number
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I apologize for any inconvenience this may cause you; however, your PIN must be verified in the automated system before I am able to share account specific information for your security.
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- Nov 2020
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www.ncbi.nlm.nih.gov www.ncbi.nlm.nih.gov
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RRID:ZFIN_ZDB-GENO-200128-7
DOI: 10.7554/eLife.60445
Resource: (ZFIN Cat# ZDB-GENO-200128-7,RRID:ZFIN_ZDB-GENO-200128-7)
Curator: @evieth
SciCrunch record: RRID:ZFIN_ZDB-GENO-200128-7
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- Sep 2020
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ecourse.auca.kg ecourse.auca.kgFYS.pdf4
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'[My] mouth [that] cursed you shall bless [you] as well!
being rude and kind in the same time.
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Come, Shamhat, 1 will fix your destiny,
It shows the confident.
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In [your presence] I will pray [to Anu,] father of the gods,
It shows that Sumerians believe in different gods.
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He has a second dream, in which he is dragged down to the Netherworld by the Angel of Death and granted a vision of hell.
It shows that Sumerians believe to the life after death.
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elifesciences.org elifesciences.org
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RRID:ZFIN_ZDB-GENO-070702-7
DOI: 10.7554/eLife.44431
Resource: (ZFIN Cat# ZDB-GENO-070702-7,RRID:ZFIN_ZDB-GENO-070702-7)
Curator: @evieth
SciCrunch record: RRID:ZFIN_ZDB-GENO-070702-7
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- Jan 2020
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www.sciencedirect.com www.sciencedirect.com
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RRID:ZFIN_ZDB-GENO-160122-7
DOI: 10.1016/j.cell.2019.01.019
Resource: (ZFIN Cat# ZDB-GENO-160122-7,RRID:ZFIN_ZDB-GENO-160122-7)
Curator: @ethanbadger
SciCrunch record: RRID:ZFIN_ZDB-GENO-160122-7
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reg.clinicalgenome.org reg.clinicalgenome.org
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allele id lookup result: https://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA915940549
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www.sciencedirect.com www.sciencedirect.com
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7
Monarch lookup result: https://monarchinitiative.org/phenotype/HP:0000717
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muscular hypotonia
Monarch lookup result: https://monarchinitiative.org/phenotype/HP:0001252
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intellectual disability
Monarch lookup result: https://monarchinitiative.org/phenotype/HP:0001249
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motor development
Monarch lookup result: https://monarchinitiative.org/phenotype/HP:0001270
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speech and language development
Monarch lookup result: https://monarchinitiative.org/phenotype/HP:0000750
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developmental delay
Monarch lookup result: https://monarchinitiative.org/phenotype/HP:0001263
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- Nov 2019
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canvas.uoregon.edu canvas.uoregon.edu
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The only place he could think of was the Trianon in mid-town
Mapping Assignment Location - Emi and Ichiro go dancing
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Thinking that he heard a knocking on the front door, he remained still and listened. It came again, faintly, hesitantly. He went through the store, wondering who it could be.
Emi coming to Ichiro's store to talk about his mother's passing - Mapping Assignment location
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He squirmed uneasily and wondered if Taro would acknowledge the telegram which he had sent the day before after finally having hunted down the information that he was taking basic training in a California camp
Mapping Assignment Location - Taro's location during Ma's funeral
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HE FUNERAL WAS HELD SEVERAL DAYS LATER AT THE BUDDHIST church up on the hill next to a playground.
Mapping Assignment Location - Ma's funeral
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They did not speak again until the car was beside the grocery store.
Mapping assignment
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I went to Portland with him.”
Mapping Assignment Location
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‘Put my ashes in an orange crate and dump them in the Sound off Connecticut Street Dock where the sewer runs out,’
Mapping Assignment Location
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It scared him to think that he might be sobering up.
What scared him more, sobering up or facing reality?
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dh101.humanities.ucla.edu dh101.humanities.ucla.edu
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We can take any data set and put it into a pie chart, a continuous graph, a scatter plot, a tree map and so on.
I have always questioned this because I feel that some information or data can not be put into any sort of visual representation.
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dh101.humanities.ucla.edu dh101.humanities.ucla.edu
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The use of a legend and symbols helps, but cartographers, artists, and designers have also introduced spatial distortions and warps that are unusual and imaginative.
I never thought that mapping would be so complex!
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how we might expand the conventions of map-making to include the kinds of experiential aspects of human culture that are absent from many conventions.
I am interested to see where the mapping industry goes, as everything is becoming digitalized, will paper maps still be used? What demographic still buys them now and who will buy them in the future?
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very projection is a distortion, but the nature of the distortions varies depending on the ways the images are constructed and the purpose they are meant to serve
I find this to be so interesting. Every map of the globe that I look at I assumed was the same, but this is making me rethink that logic.
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- Oct 2019
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www.prnewswire.com www.prnewswire.com
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Innovation in Customers' Hands at New 7-Eleven® Lab Store Retailer Celebrates New Sylvan | Thirty Location with March 22 Grand Opening News provided by 7-Eleven, Inc. Mar 27, 2019, 09:30 ET Share this article IRVING, Texas, March 27, 2019 /PRNewswire/ -- Made-to-order smoothies and agua frescas … street tacos on handmade tortillas … a growler refill station pouring local craft beers … baked-in-store cookies and croissants … patio and inside dining areas … The newest 7-Eleven® location is a lab store and an experiential testing ground, where customers can try and buy the retailer's latest innovations in a revolutionary new store format. The newest 7-Eleven location in Dallas is a lab store and an experiential testing ground, where customers can try and buy the retailer’s latest innovations in a revolutionary new store format. 7-Eleven, Inc. celebrated the grand opening of its new lab store in Dallas – and the only one in the U.S. – March 22. The store is located at the Sylvan | Thirty retail and restaurant development on Sylvan Avenue, north of Interstate 30. This location is less than two miles from the original Southland Ice House in Oak Cliff where 7-Eleven pioneered the convenience retailing concept more than 90 years ago. "Convenience retailing is light years away from the days of bread and milk being sold from ice docks in 1927, and the industry is changing at a faster rate than ever before," said Chris Tanco, 7-Eleven executive vice president and chief operating officer. "7-Eleven stays at the forefront by pushing the boundaries and being unafraid to try new things. This new lab store will serve as a place to test, learn and iterate new platforms and products to see what really resonates with customers and how we can use those learnings to influence future store designs."The lab store is also the first 7-Eleven location to incorporate the Laredo Taco Company® taqueria, and the first Laredo Taco Company location in Dallas. 7-Eleven acquired the taqueria along with Stripes® convenience stores in South Texas as part of the 1,000-store acquisition from Sunoco in 2018. Laredo Taco Company is famous in South Texas for its handmade tortillas made from scratch in stores every day as well as its popular salsa bar with on-site, daily prepared salsas, guacamole and pico de gallo. Tacos, quesadillas and plate meals include specialties not always seen in quick-serve Tex-Mex restaurants such as carne guisada, barbacoa, picadillo bistec, carnitas and breakfast tacos made with hand-cracked eggs.Some of the other innovative ideas customers will see at the new 7-Eleven lab store include: Made-to-order coffee drinks, cold-pressed juices, smoothies and agua frescas that give customers the option to customize their drinks in a full-service beverage format. Additionally, it carries novelty beverages on tap such as nitro cold brew, kombucha and organic teas. "The Cellar," an alcove dedicated to an expanded selection of wines and craft beers, with a nearby growler station that features a rotating selection of local craft beer, cider and ales on tap. At the growler station, customers can enjoy a draft of their favorite beverage with their meal onsite or fill a growler to take home. A cold treats bar with frozen yogurt, ice cream and multiple toppings Cookies, croissants and more baked-in-store daily Digital initiatives that enhance the shopping experience. Scan & Pay technology that allows customers to skip the checkout line and pay for their (non-age-restricted) purchases on their smartphones. Indoor and patio restaurant-style seating in the Laredo Taco Company portion of the store as well as bar-seating across the front windows in the retail space. Many of the new items in this 7-Eleven "innovation station" are limited-time offerings. "A lot has changed in retail and continues to change rapidly, especially the shopping experience," Tanco said. "This lab store is customer-focused and will explore new ideas that weren't even on the retail radar a few months ago."The new 7-Eleven lab store is also providing local jobs, and the company is looking for outgoing, customer-service-oriented employees for this innovative new retail-restaurant concept. To join the Sylvan | Thirty location as a sales or restaurant associate, interested people can apply online at: https://careers-7-eleven.icims.com. Under "Start your job search here," input 54716 for sales associate or 54817 for restaurant associate.About 7-Eleven, Inc.7-Eleven, Inc. is the premier name and largest chain in the convenience-retailing industry. Based in Irving, Texas, 7-Eleven operates, franchises and/or licenses more than 67,000 stores in 17 countries, including 11,800 in North America. Known for its iconic brands such as Slurpee®, Big Bite® and Big Gulp®, 7-Eleven has expanded into high-quality salads, side dishes, cut fruit and protein boxes, as well as pizza, chicken wings, cheeseburgers and hot chicken sandwiches. 7-Eleven offers customers industry-leading private brand products under the 7-Select® brand including healthy options, decadent treats and everyday favorites, at an outstanding value. Customers also count on 7-Eleven for bill payments, self-service lockers and other convenient services. Find out more online at www.7-Eleven.com, via the 7Rewards® customer loyalty platform on the 7-Eleven mobile app, or on social media at Facebook, Twitter and Instagram.SOURCE 7-Eleven, Inc.
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corp.7-eleven.com corp.7-eleven.com
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Oh Thank Heaven for 7-Eleven!®A success story fueled by customers’ needs “Give the customers what they want, when and where they want it.” Joe C. Thompson Jr. | 7‑Eleven Founder The 7‑Eleven brand is known and loved around the world, and our iconic products are a big part of the American culture. And although we’ve grown significantly over the years, our focus stays fixed on making life easier for customers. This simple idea is the reason we’re the marketplace leader. It’s also why our customers, employees, Franchisees and community leaders are proud to be part of the 7‑Eleven story. Nonstop Innovation & Customer Obsession We Did 7‑Eleven has a legacy of innovation. We were the first to provide to-go coffee cups, offer a self-serve soda fountain, operate for 24 hours a day, and yes, we even coined the phrase “BrainFreeze®” in honor of the world’s favorite frozen drink. Then came the innovation of some of our most popular menu items: the SLURPEE® drink, the BIG GULP® and then the BIG BITE®. Now, we continue our history of innovation and power it through digital initiatives. We Will From our humble beginning as the world’s first convenience store, 7‑Eleven continues its pursuit of innovative ways to cater to a new, digital-savvy generation of shoppers. As technology redefines how customers shop, we make sure to remain two steps ahead. At 7‑Eleven, we are customer-obsessed. We always poll customers to ensure we are bringing them solutions that they can’t even imagine. From offering convenient and user-friendly technology that earns our customers free products to having an ice-cold SLURPEE® drink delivered straight to their door, we are the leader in convenience and we constantly put our customers at the center of design and development. We Are It’s no secret: customers are starved for time as our world becomes increasingly connected and on the go. Our digital team strives to be at the forefront of innovation, providing effortless and convenient solutions for our customers’ needs before they even know to ask for them. We are committed to developing experiences of the future by bringing our stores to our customers wherever they are and whenever they need us. We are continuing our long-standing legacy of innovation through software and technology enhancements. We are transforming our business, one digital initiative at a time. We are redefining convenience. Learn More About Our Apps The best kind of neighbor Convenience may be our focus, but serving is our business. And that business extends beyond our stores into the communities where our customers, employees and Franchisees live, work and play. Being a great neighbor is all about investing and getting involved. It’s also about responsibility, which is one of our key business principals. That’s why we put such a focus on serving people, improving our products and protecting the planet. 7‑Eleven is proud to set the standard for responsible retailing in the convenience industry. Learn more Our Culture To lead, we serve 7‑Eleven has always been about serving the needs of our customers. This philosophy has also extended to our 7‑Eleven team, which is why our Leadership Principles play such a big part in our organization. To lead, we serve. It’s a big idea and it makes a tremendous difference in the lives of our customers, our Franchisees and our licensees. All kinds of people with one focus: making life easier We’re the convenience leader for a reason – and it has everything to do with our diverse and multi-talented team of men and women who are passionate about making life convenient. For decades – nine to be exact – we’ve counted on their unique perspectives to help us innovate and grow. They are the reasons we continue to lead in the industry. Room to move around and grow Cross-functional learning is not just allowed at 7‑Eleven, it’s applauded. We’ve got the size, stability and resources that make it possible for employees to find rewarding careers. People in all facets of our global organization discover opportunities with room to move around, try on new roles and discover untapped talents. We know that when people love what they do, everyone wins. A top-five franchisor 7‑Eleven is a brand that’s recognized worldwide. We’ve also made a reputable name for ourselves in the franchise business, and are consistently ranked as a top-five franchisor. A turnkey business model, world-class training, ongoing corporate support and special financing programs are available to increase the success rates of our Franchisees. The Store Support Center: the engine behind our stores If our stores are the fuel for busy communities, then our Store Support Center is the fuel for our stores. This is where the innovating, strategizing, forecasting, training and troubleshooting take place. From operations management and logistics to real estate, IT and human resources, our Store Support Center teams work hard to further our mission of convenience, not only for customers, but also for our Franchisees, employees and the communities we serve.
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www.franchisebusiness.com.au www.franchisebusiness.com.au
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7-Eleven moves to support animal welfare By Nick Hall | 17 Feb 2019 View comments Global convenience chain, 7-Eleven has made major changes to its supplier sourcing agreement, eliminating caged eggs for the first time. The move follows ongoing criticism of the Australian cage egg farming industry, with several brands and chains making the early decision to move to wholly sustainable sourcing in-line with welfare standards. Working closely with suppliers across all states, 7-Eleven has now ensured that only free range eggs would be available for order by all stores. Clayton Ford, General Manager Corporate Affairs, 7-Eleven said the decision to phase out cage eggs was developed in accordance with franchisee wishes, with the convenience giant announcing it will increase support to assist in the transition. “Whilst our franchisees are free to engage with alternative suppliers due to our franchise agreement, we will continue to work alongside them to encourage their involvement in this initiative by sourcing free range eggs via our centralised supply chain,” Ford said. “We recognise that this is just one step, and we will continue to review animal welfare commitments in our supply chain and 7-Eleven branded products as opportunities arise.” Want to launch a convenience store of your own? Take a look at all available franchising opportunities here.
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- Sep 2019
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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Accuracy (recovery
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- Jul 2019
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www.milestonedocuments.com www.milestonedocuments.com
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Iron Curtain
How effective was the "Iron Curtain"?
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Annotators
URL
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www.milestonedocuments.com www.milestonedocuments.com
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Romero was assassinated in 1980
His views on social and political issues was not taken well and eventually got his assassinated.
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Annotators
URL
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www.milestonedocuments.com www.milestonedocuments.com
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In the event of armed attack in Europe on one or more of the Parties to the Treaty by any state or group of states, each of the Parties to the Treaty, in the exercise of its right to individual or collective self-defence in accordance with Article 51
A "attack one, attack all" mentality
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Annotators
URL
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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Oral administration of doses
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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ROS assay
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Cytotoxicity assay by Sulphorhodamine B (SRB) me
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Cytotoxicity assay by MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5 diphenyltetrazolium Bromide)method
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Cytotoxicityscreenin
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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Test for cardiac glycosides
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Test for Terpenoids
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Tests for Flavonoids
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Tests for Phenolic compounds
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Tests for Saponins
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Tests for Tannins
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Tests for Alkaloids
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Test for Sterols
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Qualitative study of phytochemicals of N. sativadurin
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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PCR amplification
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Harvesting of cultures
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Effect of pHon growth and sporulation of the bioagent
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Effect of temperature on growth of bioagent
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Effect of different media on growth of bioagentTrichoderma
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Cultural characteristicsof bioagent Trichoderma
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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Relative distribution of virulence genes among V. parahaemolyticusfrom Cochin estuary, shrimp farm and sea food
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Production of phosphatase
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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PCR amplification of 16S rRNA gene
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DNA isolatio
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Molecular confirmation of Vibrioby 16S rRNA gene se
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Growth at different temperatures
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Growth at different temperature
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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MAR indexingand antibiotic resistance pattern amon
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sg.inflibnet.ac.in sg.inflibnet.ac.inChapter 310
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Digestion of Plasmid DNA
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Minipreparation of plasmid DNA from transformed co
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Screening of recombinant E. coli clone
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Transformation of ligation mixture in electro-competent E. coli host cells (DH5ααααstrain)
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Transformation of ligated product in chemically competent E. coli host cells (DH5αααα strain)
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Ligation reactions
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Dephosphorylation of vector
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Restriction digestion
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Ligation of eluted PCR product in pBSK vector
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Cloning of gel eluted PCR produc
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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Statistical analysis
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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PubChem Compound Database Screening
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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Assessmentof mitochondrial membrane potential(MMP or ΔΨm)
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Growth kinetic studies
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Kirby-Bauer Disk diffusion method
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Antimicrobial activity
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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. Statistical analysis
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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Lactic dehydrogenase
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Malic dehydrogenase
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Succinic dehydrogenase
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Estimation of dehydrogenases activities (SDH, MDHand LDH) in muscle and gill tissue of fish, Labeo rohita
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- Jun 2019
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krishikosh.egranth.ac.in krishikosh.egranth.ac.in
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Biological Yield(g)
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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Concentration of ideal carbon source
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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Post-vaccination declination of Transitional B cell population and increase in memory B cells in HBV positive newborns
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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Cell Counting
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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Semi dry transfer buffer
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Stripping of Ni-NTA agarose beads
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Cell transfection
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GFP-β-cateninC-C (423-634 a.a) (ARM-C)
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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Chemicals/inhibitors
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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Sonication of bacteria
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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Cytotoxicity assay
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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ELISA dilution buffer
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Lymphokines Assay
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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Gene cloning
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Large scale plasmid extraction
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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Separation ofCurcuminoids
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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Estimation of Stevioside
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Extraction from the plant material
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Extraction from the plant material
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Extraction from the plant material
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Qmmtification of stevioside
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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Mesomorphic endomor~:
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Humerus
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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The semisynthetic a globin was purified from the mixture by CM52 urea chromatography as explained below. The lyophilized sample was dissolved in 0.005 M phosphate buffer (pH 6.9) containing 8 M urea and 0.05 M 2-mercaptoethanol at a concentration of I 0-15 mg/ml and loaded onto a CM52 column (16 em x 1.5 em) equilibrated with the same buffer. After an initial wash with the same buffer, two linear gradients of(a) 100 ml each of0.005 M to 0.03 M and (b) 100 ml each of0.02 M to 0.05 M phosphate buffer (all buffers contained 8 M urea and 0.05 M 2-mercaptoethanol, and were adjusted to pH 6.9) were employed at a flow rate of 45 ml/h to elute the semisynthetic a globin. The column was finally washed with 0.05 M buffer to elute unreacted a31-141 fragment from the column. The elution profile was monitored at 280 nm. The fractions for semi-synthetic a globin were pooled, extensively dialyzed against 0.1% TF A and lyophilized. The semisynthetic yield of the protein varied between 35% to 45%
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Purification oftlte semi-!)yntltetic a globin
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- May 2019
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oll.libertyfund.org oll.libertyfund.org
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Phlegyas
Phlegyas is the son of Mars (god of war) who became outraged after the god Apollo raped his daughter. Out of rage, he set fire to the temple of Apollo. Phlegyas is in The Inferno because Virgil uses him to represent the sins of wrathful and sullen. In The Inferno, Phlegyas is responsible for Dante and Virgil across the Styx in his boat.
Tags
Annotators
URL
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shodhganga.inflibnet.ac.in shodhganga.inflibnet.ac.in
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Quantitative screening for determination of xylanase in shake flask
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Sonicated cells of E. coli having recombinant vector was centrifuged. Supernatant was dispensed into 0.2 % v/v xylan agar plate and incubated for 4 h. The plates were then flooded with Congo red solution (0.2 % w/v) for 30 min and destained with 1M NaCl solution till a clear zone of xylan hydrolysis was visible. The plates were gently shaken on a shaker to accelerate the process of staining/destaining
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Qualitative detection of xylanolytic activity by plate assay
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DETECTION OF XYLANASE ACTIVITY
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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All site-directed mutagenesis studies were performed usmg the QuickChange mutagenesis kit (Stratagene) following the manufacturer's instructions. It is a PCR based method for introducing point mutations, replace amino acids and delete or insert single or multiple amino acids into desired plasmid constructs. Primers containing mutations were designed and PCRs were performed using "wildtype" construct as template. The PCR product was' subjected to digestion with DpnI endonuclease, which is specific for methylated DNA. Following DpnJ digestion, the parental DNA template gets cleaved and DNA containing desired,mutation is selected. The residual mutant nicked DNA was transformed in E. coli DH5a competent cells and the resulting plasmids were isolated and sequenced to confirm incorporation of the desired mutations.
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ite directed mutagenesi
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Parasite culture was used to make a thin blood film on a glass slide. After air drying, the thin smear was fixed in methanol for about 30s. A fresh 5 to 10% , giemsa solution was prepared in phosphate buffer (list I). The slide was placed in a I staining jar and the giemsa solution was poured on the slide for 20 min and' subsequently rinsed thoroughly under running tap water. The stained parasites, were then observed under a light microscope using 100X objective.
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Giemsa staining o/thin blood smear o/parasite cultures
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sg.inflibnet.ac.in sg.inflibnet.ac.in
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cold PBS followed by incubation with fluorophore labeled secondary antibody at appropriate dilution for 30 min at 4°C. The fluorescence was then visualized under a fluorescence microscope or analyzed by flow cytometry
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Immunostaining in Live cells: Immunostaining on live cells was performed by harvesting and resuspending cells in ice-cold PBS. The cells were then incubated with an appropriate dilution of the primary antibody for 1 h at 4 °C following which two washes were given with ice-
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Immunostaining in fixed cells: The cells were fixed with 4% formaldehyde for 20 min, following which two washes were given with ice-cold PBS. Permeabilization and blocking were performed simultaneously by incubating the formaldehyde fixed cells in PBS containing 0.1% saponin and 3% normal goat serum for 30 min. The cells were washed once with ice-cold PBS. The permeabilized cells were incubated with the primary antibody at an appropriate dilution for 1 h at room temperature following which three washes with ice-cold PBS was given. These cells were then incubated with fluorophore conjugated secondary antibody (IgG) for 1 hat room temperature following which three washes with ice-cold PBS were given. The nuclei were stained with Hoechst 33342 at a concentration of 1 Jlg/mL for 2 min at room temperature. The staining was then visualized under a Nikon TE2000E fluorescence microscope using appropriate filter blocks. Image acquisition was carried out using a high-resolution Retiga Exi camera (Q-imaging, Surrey, BC, Canada) and subsequent image analysis was performed on Image-Pro Plus software v5.5 (Media Cybernetics, Silver Spring, MD). Alternatively, the fluorescence staining was detected by flow-cytometry (BD-LSR, Beckton Dickinson, NJ, USA) using an air-cooled argon ion laser (488 nm) at appropriate florescence channels. Subsequent data analysis was performed on WinMdi software (Microsoft, v 2.9)
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Immunocytochemistry
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checks the stereochemical quality of protein structures. The output of the program consists of comprehensive listings of the stereochemical parameters. These files were analyzed to determine if the error in the various stereochemical parameters were within acceptable limits. If any parameter ofthe model was found to be outside the accepted range, they were corrected by adjustment of the concerned residue followed by refinement. The elbow angles of all the Fab structures were calculated using web based applet developed by Standfield et a/ (Stanfield et al., 2006). The buried surface areas (i.e. the area rendered inaccessible to a 1.4 A sphere) were determined using PISA web server (Krissinel and Henr:ck, 2007). CONTACT program of the CCP4 package (Elizabeth Potterton, 2003) was used to determine van der Waals contacts and hydrogen bonds between peptide and Fab. Vander Waals contacts were defined to be present between atoms if they were within 4 A of each other. Hydrogen bonds were assigned for donors and acceptor atoms when the distance between them is less than 3.5 A. In case of hydrogen bonds where the nitrogen atom is the donor, the N-H ... O angle should be greater than 120°. When an oxygen atom is the donor, a cut off value of 90° was used. All Fab-peptide complexes ofmAbs; BBE6.12H3 and 36-65 were compared in terms of various parameters such as elbow angle, peptide and CDR conformation, buried surface area, van der Waal contacts and hydrogen bonding. To compare conformations, RMSD in the position of the Ca as well as all atoms were calculated using SUPERPOSE program of CCP4 package (Krissinel and Henrick, 2004). The CDR conformations in the liganded and unliganded forms were also compared.
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Deviations from ideal geometry of the various structures were analyzed using PROCHECK (R. A. Laskowski, 1993) from the CCP4 suite. PROCHECK
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Validation, analysis and comparison of models
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of the plasmid DNA with 0.5 volume of cold isopropanol. The mixture was kept on ice for 10 min. and centrifuged at 12,000 rpm at 4°C for 15 min. DNA pellet thus obtained was washed with 80% ethanol, dried and dissolved in 50 J.!l TE buffer (pH 8.0). Minipreps were screened by restriction digestion. 5 J.tl of plasmid DNA was incubated with 5 units of appropriate enzyme(s) and 150 units of RNaseT1 for 2 h and the products were analyzed on an agarose gel to identify the positive clones.
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5 ml LB containing 100 J.tg/ml of ampicillin was inoculated with single. bacterial colony picked from the culture plates. The culture was grown for 12 h at 37 °C with vigorous shaking. Cells were harvested from 3 ml of culture by centrifugation at 3000 rpm in a microfuge (Plastocraft) at 4 °C for 15 min. Added 200 J.tl of TEG buffer was added to the cells, and tt.ey were gently resusupended to get a uniform suspension and kept on ice for 5 min. 400 J.tl of freshly prepared alkaline-SDS solution was added to the cell suspension and mixed well by inverting the tubes followed by an incubation on ice for 10 min. Subsequently, 300 J.ll of chilled potassium acetate solution was added and mixed thoroughly by vortexing. The mixture was centrifuged at I 0.000 rpm at 4 °C for 15 min.. The supernatant was collected and phenol-chloroform extraction was performed followed by precipitation
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Mini Plas~id Preparation and Screening
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Progesterone levels were estimated from sera of bonnet monkeys which were bled biweekly using a radioimmunoassay employing reagents and protocol as prescribed by the W.H.O. Matched Assay Reagent Programme (Sufi et al., 1983). Each sample was run in duplicates. Progesterone was extracted from serum (0.1 ml) by the addition of 2 ml of ice-cold ether in each tube and vortexing for 2 min. The tube was immersed in liquid nitrogen in order to flash freeze the serum phase and the unfrozen ether phase which contained the extracted steroid hormone was decanted into another tube. The ether was allowed to evaporate 0/N and 0.5 ml of steroid assay buffer (0.1 M PBS, pH 7 .3, 0. 1% thiomersal and 0.1% gelatin) was added to the tubes and the tubes were incubated at 40°C for 30 min. Steroid sticking to the walls of the tubes was recovered by vigorous vortexing. 100 J..LI of anti-progesterone Ab (at a dilution giving -50% binding of tritiated p.rogesterone in the absence of unlabelled competing progesterone) was then added to the tubes followed by addition of 0.1 ml of 3H-progesterone ( -10,000 cpm/tube). The mixture was incubated for atleast 16 hrs at 4oc. Unbound progesterone
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was separated by addition of 0.2 ml of ice cold assay buffer containing 0.625% activated charcoal and 0.0625% dextran and incubated for 30 min at 4oc. This was followed by centrifugation at 2500 rpm for I 5 min at 4°C. The supernatant was carefully decanted into scintillation vials and 4 ml of scintillation fluid (0.4% 2,5 diphenoxazole; 0.01% POPOP [1-4 bis(5-phenyl-2-oxazolyl)benzene] in sulfur free toluene) was added and counted in a liquid scintillation beta counter (Beckman Instruments, California, USA). The amount of progesterone per ml of serum was calculated from a standard curve with known amounts of progesterone in each assay.
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Progesterone Radioimmunoassays
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Expression conditions for bZP3 under the polyhedrin promoter were standardized using the Northern blot and Western blot analysis of cells infected with the VI virus. Sf9 cells, seeded at a density of 1.5 million in a 35 mm petridish were allowed to attach for I h at 27oc. The medium was removed and the cells were infected with AcNPV (Autographa californica nuclear polyhedrosis virus) or VI at -10 MOl for 1 h. The infected cells were harvested at different time points from 0-84 h pi. The cells (-2X I o6) were washed with chilled PBS and resuspended in I ml of denaturing solution ( 4 M GITC, 25 mM sodium citrate, pH 7, 0.5% sarcosyl, and 0.1 M BME) followed by addition of 50 Jll of 2 M sodium acetate (pH 4) and 500 Jll water saturated phenol and 1 00 Jll chloroform:isoamyl alcohol ( 49: 1 ). The suspension was mixed thoroughly after the addition of each reagent, vortexed for 1 0 sec and cooled on ice for 15 min. The aqueous and the phenol phases were separated by centrifugation at 12,000 rpm for 20 min in a refrigerated microfuge. The aqueous phase was transferred to a fresh tube and 500 Jll isopropanol was added. RNA was precipitated at -20°C for 1 h, and pelleted at 12,000 rpm for 20 min at 40C. The RNA pellet was dissolved in 300 Jll denaturing solution followed by addition of 300 Jll of isopropanol. RNA was reprecipitated at -2ooc for 1 h, washed with 75% ethanol and the pellet collected by centrifugation at 12,000 rpm in a refrigerated microfuge. RNA was dissolved in 25 Jll of 0.5% SDS by heating at 65°C for 10 min and stored at -700C. RNA was quantitated and 5 Jlg of RNA corresponding to each time point was resolved on a 1.2% agarose formaldehyde gel, transferred to a nylon membrane and probed with 32p labeled bZP3 probe. Cells harvested at different time points from -2X 106 cells 12-84 h pi were pelleted down, washed with 10 mM PBS, pH 7.4, and lysed in reducing buffer and resolved on a 0.1% SDS-10% PAGE as described earlier. The supernatant was concentrated to lOX for loading on the gel.
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Expression 'Of bZP3 in BEVS
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Double stranded plasmid pBluescript-bZP3 DNA was sequenced using Sanger's dideoxy chain termination method (Sanger et al., 1977) using the Sequenase version 2.0 kit according to the protocols recommended by the manufacturer. Purified plasmid DNA (5 J..Lg) and 2 pM of the sequencing primer was used in the sequencing reaction. Table 2 gives a list of the primers used for sequencing of the bZP3 eDNA clones. bZP3 sequence was confirmed by sequencing three independent clones 401, 403 and 404.
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Sequencing of bZP3
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minigel alongwith unligated vector to test the ligation. The ligated DNA was used to transform competent ~.coli cells.
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Wherever possible, the cloning of DNA fragments was achieved by ligation of compatible sticky ends generated on the vector as well as the insert by digestion with the same enzyme. Self ligation of the linearised vector with compatible sticky ends was minimised by dephosphorylation of the vector DNA using bacterial alkaline phosphatase. The ligation conditions for each batch of T4 DNA ligase were standardised using Hind III generated fragments of lambda DNA as a test sample for sticky end ligation. Routinely, 200 ng of vector DNA was mixed with 2 - 5 fold molar excess of the insert fragment DNA, 2 ul each of the 10 X ligase buffer 500 mM Tris. HCl, pH 7. 5, 100 mM Mgcl2 ) , 10 mM ATP, and 200 mM DTT. The final reaction volume was adjusted to 15 - 2 0 ul with sterile double distilled water, and 0.5 - 1 ul of T4 DNA ligase ( 103 units I ml ) was added. The contents were mixed well and incubated at 13°C for 12 -16 hours. An aliquot of 2 ul was electrophoresed on a
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Ligation of DNA fragments.
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Intensifying screens were from Kiran X-ray Screens, India. X -ray films were from Agfa -Gevaert, Belgium, Kodak, USA, or Hindustan Photo Films, India. Developer and fixer were from Hindustan Photo Films, India.
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Materials for autoradiography.
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separately on LB plates containing 100 j...tg/ml ampicillin, 80 j...tg/ml of X-gal and 20 mM of IPTG. The plates were incubated at 37°C for 12 h.
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The DH5a strain of E. coli was grown overnight (0/N) in LB at 37°C and subcultured ( 1: 1 OO)in 100 ml of fresh LB. The culture was grown until absorbance at 600 nm (A6oo) reached 0.4. The culture was centrifuged at 2500 X g for 15 min at 4°C. The cell pellet was resuspended in 10 ml of freshly prepared sterile ice cold CaC}z (100 mM) solution and incubated for 30 min on ice. Cells were centrifuged at 1800 X g and the pellet was very gently resuspended in 2 ml of chilled CaCh (100 mM) containing 15% glycerol. Aliquots of 100 111 were dispensed into sterile, chilled 1.5 ml eppendorf tubes and stored at -70°C until further use. For transformation, the ligation products from the above reactions were added separately to a vial each of DH5a competent cells thawed on ice. The contents were gently mixed and incubated on ice for 30 min. The cells were then exposed to heat shock at 42°C for 90 sec and incubated on ice for another 2 min. The transformed cells were grown in 1 ml of LB medium for lh at 37°C with shaking for the expression of the ampicillin resistance marker gene W-lactamase). Aliquots from each transformation were plated
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Preparation of competent cells and transformation
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Thecapabilityofheadkidneyneutrophilstomovewasassayedbyamigration-under-agarosetechniquemodifiedfromNelsonetal.(1975).ThemethodhasbeendescribedbySaloetal.(1998).Thedistance,thecellshadmigratedfromthemarginofthewelltowardsthewellcontainingcasein(directedmigration)andintheopposite direction(randommigration)weremeasuredunderthemicroscope.
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Migration
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Thereactionproduction,p-nitrophenolinacidphosphatewasmeasuredspectrophotometricallyat415nmagainstreagentblank.Theenzymeactivitywascalculatedfromthestandardcurveandexpressedasmicromolesofp-nitrophenolformedperhourpermilligramprotein.Therateofhydrolysisofp-nitrophenolphophateisproportionaltotheenzymepresentinthetissue.p-nitrophenylphosphate+NaoH—phosphat?-—>p -nitrophenol+phosphateThecolordevelopedinalkalinephosphataseactivitywasreadat410nmagainstreagentblankspectrophotometrically.Theactivityoftheenzymewasexpressedaspmolphenolformedmin'1mg'1protein
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ThealkalinephosphataseactivitywasestimatedbythemethodofMorton(1955)usingp-nitrophenylphosphatesocolorlessinsolutionbutuponhydrolysis,thephosphategroupliberatesp-nitrophenylwhichishighlycoloredinalkalinesolution
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Alkalinephosphatase(AKP:ortho-phosphoricmonoester-phosphohydrolase;E.C.3.1.3.1)
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SamplesofC.punctataweretakenfromtheacclimationtanksandslightlyblottedonpapertowelstoremoveexcesswater.TheywereplacedindividuallyonaWhatmanfilterpaperinaonelitreglassbeaker.Theindividualswereimmediatelytransferredtobigglassdesiccatorscontaining250mlofthefollowingsolutionsfordesiredhumiditylevelsusinggradedsolutionsofKOHasdescribedbySolomon(1951).Watergiving95to97%relativehumidity(RH),mean95%;sodiumchloridegiving72to76%relativehumidity,mean75%;calciumchloridegiving28to31%relativehumidity,mean35%.Thedesiccatorswereplacedinanincubatorataconstanttemperatureof28°±1°Cwithdeterminationsofsurvivalandbodyweightatregularintervals.Thecontainerswereweighedatintervalstothenearest0.1mgandweightlosswasassumedtoequalwater loss
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Survivaloutofwater
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dried. These were counted directly to determine the total counts. In duplicate tubes, 1pl of the diluted probe was added to 100pg of carrier nucleic acid (tRNA or Herring Sperm DNA) in a total volume of 100pl. To this 500pl of ice-cold 5% TCA was added, mixed thoroughly and incubated on ice for 15-20 min. Glass fiber filters were wet (in duplicate) properly with 5% TCA and then these samples were applied on to them under vacuum. The filters were washed twice with 5ml of chilled 5% TCA and then air dried after rinsing with 2m1 of acetone. All the dry filters were inserted into scintillation vials containing scintillation fluid and the counts were taken in a liquid scintillation a-counter (LKB Wallac, 1219 Rackbeta, Sweden). The percentage incorporation, specific activity and the total amount of RNA made was then calculated according to the standard procedures. % incorporation =Incorporated cpm X100 Totalcpm Total RNA made (ng) = % incorporation X 338 Specific activity of probe = Total cpm incorporated p.g of RNA synthesized
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To determine the percentage of incorporation and probe specific activity, 1:10 dilution of the labeled probe was made in NFW. lpl of this was spotted on to duplicate glass fiber filters (Whatman GF/ A, U.S.A.) and ai
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richloro acetic acid (TCA) precipitation:
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For adherent cells from which lysates have to be prepared , culture medium was removed and cells were washed with ice cold 1X PBS twice and then scraped with cell scraper in Cell Lysis buffer. Cells were rotated at 4°C for 30min at cold room and centrifuged at 13000 rpm for 10min at 4°C. The supernatant was collected and protein concentration was estimated using BCA assay. For standard western, 50-70μg of protein was loaded on to the gel
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Lysate Preparation for Immuno- blotting
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d. 3 μl of sample prepared in low stringency buffer was added to the spot activated with low stringency buffer and incubated in humid chamber for 30 min. and removed using whatman strips. (same protocol was repeated for the samples prepared in high stringency buffer on spots activated with high stringency buffer). e. Stringent washings were given to each spot with 5 μl of low stringency buffer/ high stringency buffer/ buffer of pH 3.0/ pH 5.0/ pH7.0 for 30 sec and removed using Whatman filter paper strips. f. 1-2 μl of SAP matrix was added to each spot and allowed to dry. g. The chip was then placed in the SELDI machine
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One set of cell extracts was prepared in low stringency buffer by mixing cell extracts and low stringency buffet in 1:1 ratio and another in high stringency buffer. b. 10 μl of low stringency/high stringency buffer was added to the spots on the chip and incubated in a humid chamber for 5 min. c. Buffer was removed using Whatman strips without touching the spot surface. This step was repeated once
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Activation of LSAX (strong anion exchange ) array
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Overnight cell culture raised in LB medium was subcultured 1:100 in LB with 20 mM MgCl2. When the A600 reached 0.4-0.6, the culture was centrifuged at 2800g for 5 min at 4 ̊C. To the cell pellet 0.4 volumes of ice-cold TBF-I buffer was added and incubated on ice for 15 min. The cell suspension was centrifuged at 2800g for 5 min at 4 ̊C and the cells recovered were dissolved in 0.04 volume of ice-cold TBF-II buffer and kept on ice for 45 min. 100 μl aliquots of these competent cells were used for transformation using the normal transformation protocol
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TBF method for preparation of high competency cells
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For routine plasmid transformations, where high efficiency is not required, the following method which is a modification of that described by Sambrook and Russell (2001) was used. An overnight culture of the recipient strain was subcultured in fresh LB and grown till mid-exponential phase. The culture was chilled on ice for 15 min, and the steps hereafter were done on ice or at 4°C. The culture was centrifuged, and the pellet was resuspended in one third volume of cold 0.1 M CaCl2. After 15 min incubation on ice, the cells were again recovered by centrifugation, and resuspended in one tenth volume of cold 0.1 M CaCl2. The suspension (0.1 ml) was incubated on ice for 1 h after which DNA was added (~10-100 ng of DNA in less than 10 μl volume). The mixture was again incubated on ice for 30 min, and then heat shocked for 90 seconds at 42°C. Immediately 0.9 ml of LB broth was added to the tube and incubated at 37°C for 45 min for phenotypic expression of the antibiotic marker before being plated on selective medium at various dilutions. A negative control tube (with no plasmid DNA addition) was also routinely included in each of the experiments
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Calcium chloride method
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Transformation protocols
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Antibiotics were used at the following final concentrations (μg/ml): Rich media Minimal media Ampicillin (for plasmids) 100 50 Ampicillin (chromosome) 30 30 Chloramphenicol (for plasmids) 50 25 Chloramphenicol (chromosome) 25 25 Kanamycin 50 25 Nalidixic acid 50 - Rifampicin 100 - Streptomycin 50 100 Streptomycin 100 200 Spectinomycin 50 100 Tetracycline 15 8 Trimethoprim (for plasmids) 60 30 Chloramphenicol 0.1mg/ml The 10 mg/ml chloramphenicol stock in ethanol was used to make 0.1mg/ml solution in water
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Antibiotics
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were then centrifuged at 1065 x g for 5 min at 4°C. The pellet obtained was resuspended in 1mL ice cold PBS and washed 5 times with PBS. Finally the pellet was resuspended in 1mL lysis buffer (10mM Tris-HCl containing 0.1% Triton X-100) and fluorescence was measured at 380nm/525nm. To normalize different samples and account for any errors in cell number between samples, 10011L of the above sample was also used for protein estimation, carried out as described above
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Monodansylcadaverine (MDC) is a selective marker for autophagic vacuoles (Biederbick et al., 1995). It is an auto-fluorescent drug accumulates in acidic compartments by ion trapping and also is thought to interact with the membrane lipids of the vacuoles. Stock MDC (50mm, prepared in acetic acid) was diluted to a concentration of 50!lM in M199 medium containing 10%FBS. 107 parasites after appropriate treatment were resuspended in 1mL of working stock and incubated in the dark for 10 min at RT. These
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Assay for measuring the level of autophagy in Leishmania parasites (MDC staining)
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DNA fragments were resolved on 1-2 % agarose gel containing 0.5~-tg/mL ethidium bromide in Tris-Acetate-EDTA (TAE) buffer (40mM Iris-acetate, 2mM EDTA, pH 8.1). The samples were mixed with equal volume of 2X loading dye containing bromophenol blue, and the samples resolved by applying a voltage of 5-7 V /em. The resolved DNA fragments were visualized under ultraviolet illumination (312nm) and the relative band size was determined by comparison against DNA ladder with bands of known sizes. When required the images were acquired using a UVP Gel Documentation system
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Agarose gel electrophoresis
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5% glycerol)containing (i) 5′-end-labeled DNAfragmentof 1200 cpm radioactive count(ii) 1 μg each of bovine serum albumin andpoly(dIdC)(iii) the protein at the indicated monomer concentrations and (iv) when required,co-effectorsat specified concentrations. The reaction mixture was incubated at room temperature for 30-minsand the complexes were resolved by electrophoresis on a non-denaturing 5%polyacrylamide gel (39:1 acrylamide:bisacrylamide)in 0.5X TBE buffer pH8.3, at 12.5V/cm for 3 hrs at 18°C.The gels were then dried on a gel drier at 80°C for 45 minsand the radioactive bands were visualised with a Fujifilm FLA-9000 scanner.For DNA bending EMSA, co-effectors were not added in the binding reaction but at aconcentration of 0.1 mM in both the gel and running buffer
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The DNA templates were obtained by PCR from E. coligenomic DNA. After 5-end labeling, the PCR fragments were purified by electroelution following electrophoresis on 6% native polyacrylamide gels (Sambrook and Russell,2001). EMSA reactions were performed in 20 μl reaction volume inEMSA binding buffer(10 mM Tris-Cl at pH 7.5, 1 mM EDTA, 50 mM NaCl, 5 mM dithiothreitol, and
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Electrophoretic mobility shift assay (EMSA)
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Typically 200-300 ng of DNA was used in each ligation reaction. The ratio of vector toinsert was maintained between 1:3 to 1:5 for cohesive end ligation and 1:1 for blunt endligation. The reaction was generally performed in 10 μl volume containing ligationbuffer (provided by the manufacturer) and 0.05 Weiss unit of T4-DNA ligase, at 16ºCfor 14-to 16-hrs. On using the rapid ligation kitfrom Fermentas, incubation was at 22ºC for 1-2 hrs
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Ligation of DNA
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purification of DNA fragments werefrom Qiagen or HiMedia. The oligonucleotide primers used in this study were mainly synthesised by Ocimum Biosolutions or MWG Biotech. The radioactive chemicals were procured from BRIT Mumbai
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Chemicals were obtained from commercial sources. Most of the chemicals such as amino acids, antibiotics, sugars, IPTG, ONPG and X-gal were obtained from Sigma Chemical Co. The media components for the growth of bacteria were mostly from HiMedia laboratories. The materials used in the recombinant DNA experiments such as restriction endonucleases, T4-DNA ligase, DNA-polymerases and DNA size markers were obtained from companies including New England Biolabs, MBI Fermentas and Stratagene.RNA isolation chemicals like Reverse transcriptase, trizol, RNA loading buffers and dyes and RNA size markers were obtained from Invitrogen and Sigma. Protein markers were obtained from MBI Fermentas. Kits for plasmid isolation,
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Chemicals
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Log-phasecells grown in YPD medium containing or lacking CaCl2and FK506 were collected, PBS-washed and loaded with ratiometric, high affinity, membrane-permeable calcium indicator, Fura-2 AM (10 M; Sigma #47989). After 30 min incubation at 30◦C, labelled cells were washed thrice with cold PBS, suspended in PBS and fluorescence was recorded at 505 nm with dual excitation at 340 and 380 nm. The ratio of fluorescence intensities between 340 and 380 nm, representing Ca-bound and Ca-free Fura-2 molecules, respectively, reflected free intracellular calcium concentrations
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Measurement of intracellular calciumlevels
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the disrupted gene, BLAST N of the sequences from rescued plasmids was performedagainstC. glabrataGenolevures database (http://www.genolevures.org/blast.html
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Identification of disrupted locus in Tn7insertion mutants was carried out as described previously(Kaur et al.,2004). Disrupted locus in each mutant is physically marked with a mini Tn7 transposon derivative containing conditional origin of replication R6K(facilitates Tn7recovery), S. cerevisiae URA3and Klebsiella pneumoniae hphgene (confers resistance to hygromycin B)(Castaño et al.,2003). Briefly,genomic DNA was isolated fromovernight grown Tn7insertion mutants using spheroplast lysis method. After RNAse treatment, 10μgDNA was either digested withMfe1or SpeIrestriction enzymeas the Tn7 cassette lacks these enzyme sites. Followingovernight digestion, DNA wasprecipitated with 1ml ethanol and 1/10thvolume of 3 M sodium acetate (pH 5.2).DNApellet was washed twicewith ice-cold 70% ethanol, air driedand resuspendedin sterile MQ water. DNA was recircularized withT4 DNA ligase. Resultant circular plasmid contains the Tn7cassette flanked on either side by the gene,it has disruptedin the genome of C. glabrata.This circular plasmid DNA was transformedin E.coliBW23473 strain,which contains protein Π (the product of the pir gene)required by R6Korifor replication.Transformation of circularized DNA in E.coliBW23473 electrocompetent cells was performedas described below.Plasmids fromselected transformants were isolated and sequenced with outward primers from Tn7right and left ends to sequencethe disrupted gene fragment.For identification of
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Tn7insertion mutant rescueand gene identification
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E. coli DH5α ultra-competent cells were transformed with plasmid DNA by heat shock at 42 ̊C for 90 sec as described previously in Molecular Cloning-A Laboratory Manual (Sambrook and Russell,2001). Bacterial transformants were selected on LB agarmediumcontaining appropriate antibiotics. Transformants obtainedwere colony purified on LB plates containing antibiotics.Presence of the desired insertwas first verified by colony PCR followed by PCRusing extracted plasmid DNA as template
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Bacterial transformation
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activated TLC silicagel-60plate and transferred to theTLC chamber. After the solvent had migratedupwards (1.5 cm fromthetop), TLC plate was removed, air dried behind perspex shield, wrapped with cling plastic wrap and was exposed to phophorimager screenfor 2 h. Phosphorimager screen was scanned usingaFugi-FLA 9000 scanner
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To resolvephospholipids,a TLC chamber was prepared by pouring50 ml developing solution and sealing the chamber with aluminium foil so that developing solution can generate vapor. TLC silicagel-60plate(Merck)was incubated at 80ºCfor 4 h for activation. After 30 min of TLC chamber preparation, phospholipidsextracted from C. glabratacells werespotted at thebottom (1.5 cm fromthelowerend) of the
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Seperation of phospholipids by thin layer chromatography(TLC)
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PI-3kinase reaction was set up ina total volume of50μlin a 1.5 ml microcentrifuge tube as described below.PI-3 kinase reaction buffer = 25 μlSpheroplast lysate = 20 μl (equivalent to 10 μg protein)Sonicated phosphatidyl inositol = 5 μlReaction mix was incubated at 25ºC for 20 min and enzyme reaction was stopped by adding 80μlHCL (1N) solution. To extract phospholipids, 160 μl chloroform:methanol (1:1) was added to the reaction mix withcontinuous mixing. Organic phase containing phopholipidswas separated fromaqueous phase by centrifugation at 7,500g for 4 min at 4ºC and transferred to a new vial. Using vacuum evaporator apparatus, solvent was evaporated and phospholipidsweredissolved in 10 μl chloroform
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PI-3 kinase reaction set up and phopsholipid extraction
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10 mg phosphatidylinositol-sodium salt(from Glycine max)was dissolved in 2 ml chloroform to prepare a 5 mg/ml stock solution. This solution was prepared in a small glass vial aschloroformis known to reactwith polypropylene. Small aliquots of stock solution were madeand stored at -20ºC till further use. To avoid spillage due to vapor pressure, vials containing phosphatidylinositol-sodium salt solutionwereopened very carefully.To prepare sonicated phosphatidylinositolfor one PI-3 kinase reaction, 2 μlof the stock phosphatidylinositolsolution (10 μg) wastransferredtoanew1.5 ml microcentrifuge tube. Using vacuum evaporator apparatus, chloroformwas evaporated from the solution and phosphatidylinositol-sodium saltwas resuspended in 5 μl sonication buffer.For sonication, a total of 20 pulses, each of 30 sec with30 sec resting time weregiven on ice
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