One must be able to add one's own private links to and from public information. One must also be able to annotate links, as well as nodes, privately.

Sau giai đoạn giảm sốc vừa qua, có nhiều NĐT đã bị thua lỗ nặng nề. Đây là điều đáng buồn, nhưng chúng ta nên bình tâm lại. Tôi xin chia sẻ một số quan điểm về giai đoạn hiện nay:1. Đáy thường chỉ nhận biết khi đã qua, nhưng chúng ta phải nhận ra, chứ không nên bảo thủ 1 chiều. Mốc 1157 là đáy 1, mốc 1203 có thể là đáy 2. Cả 2 mốc này đã được confirm.2. Thị trường đã bình tâm trở lại, không chỉ là vấn đề tâm lý, mà cả bao gồm cả về những vấn đề khác như trạng thái Margin, short sell, ...3. Thanh khoản giai đoạn tới sẽ không còn như trước vì lý do dòng tiền rút ra (một phần từ Trái phiếu DN, một phần tiền SX-KD, một phần do thua lỗ, một phần do quá sợ CK, ...). Nhưng nếu quan sát kỹ, chúng ta có thể nhận thấy mức 10k-11k tỷ / phiên là đáy thanh khoản. Kịch bản có xác suất cao trong thời gian tới là tăng dần đều, ổn định ở mức 14k-15k tỷ/ phiên.4. Vĩ mô thế giới có nhiều bất lợi, nhưng vĩ mô trong nước lại không quá bi quan. Gói hỗ trợ LS được tung ra kỳ này khá lớn, hy vọng có thể giữ ổn định TT về khía cạnh dòng tiền. Bên cạnh đó, giải ngân đầu tư công cũng kỳ vọng thúc đẩy tăng trưởng để GDP năm nay vẫn đạt mức 7%. Đặc biệt, dù siết chặt hơn nhưng vấn đề Trái phiếu DN, nợ xấu ngân hàng vẫn có chiều hướng "ném chuột không làm vỡ bình". Cho nên chỉ cần TT Mỹ đừng quá xấu (chẳng hạn DJ đừng thủng 30k), thì Vn-index hoàn toàn phù hợp với điểm 13xx. Nếu dòng tiền mạnh hơn (chẳng hạn lên lại 20k tỷ / phiên), thì rật có thể sẽ chinh phục được 14xx. Rõ ràng, nội tại vĩ mô được định giá lại ở mức 1200-1400 là phù hợp, chứ không còn câu chuyện 1600-1800 cho năm nay được, nhưng cũng rất khó xuyên thủng 1200.5. Với thị trường bình ổn hơn, sẽ không còn câu chuyện "ăn bằng lần" hay "lỗ sấp mặt" nữa. Sẽ có từng giai đoạn các nhóm ngành mạnh yếu khác nhau. Trong giai đoạn này tôi ưu tiên nhóm bank, vận tải biển, bán lẻ, năng lượng. Tôi sẽ bỏ qua các nhóm có tính chu kỳ như thép, phân bón, ck. Còn một số nhóm ngành tương đối khó hơn như bđs, kcn, thủy sản, hóa chất, tôi sẽ hành xử rất thận trọng.188Like

Trong kỳ báo cáo gần nhất về chỉ số CPI của Mỹ đã có nhiều ý kiến cho rằng đã tạo đỉnh ở mốc 8.3%. Có nhiều khả năng khi báo cáo mới sẽ ra khoảng 10 ngày nữa sẽ nhìn thấy con số giảm rõ rệt. Dù ở VN luôn có độ trễ nhất định, nhưng khả năng CPI của VN cũng vẫn trong tầm kiểm soát, khoảng 5.0%-5.5% cho năm nay. Còn về Vn-Index thì rõ ràng sự điều chỉnh mạnh về đến dưới 1200 vì nhiều lý do khác nhau, nhưng có lẽ cũng đã phản ánh đầy đủ những gì xấu nhất. Việc TT có tăng trở lại hay không là khó dự báo, nhưng có thể tự tin rằng vùng 1200 đã confirm là đáy của Vn_index trong năm 2022. Còn sẽ tăng đến bao nhiêu, khi nào tăng, tăng ở dòng nào, lại là câu chuyện cần sự tư vấn chuyên sâu hơn.62Like

Trong ngắn hạn mọi dự báo đều có xác suất sai rất cao, tuy nhiên cá nhân tôi vẫn duy trì các dự báo này dựa trên cơ sở lập luận của mình. Không hẳn để hô hào (vì chả để làm gì), để câu view vì tôi không cần điều đó, cái chính là vì quá đam mê và luôn sống trong nhịp đập của thị trường. Có nhiều người lôi các tus cũ của tôi đã từng ngợi khen VCI, TCB, HPG, ra để bêu riếu, hoặc giả lôi những nhận định lạc quan của tôi về Vn-Index lên 1600, 1800 trước đây để "dìm hàng". Thực lòng mà nói, tôi cũng rất đau lòng khi những nhận định của mình gây ra những điều chưa tốt cho mọi người, trong đó có cả bản thân tôi. Nhưng bên cạnh đó, tôi nghĩ rằng đó là cuộc sống, đó là sự vận hành của thị trường mà chúng ta luôn vui mừng học hỏi từ những thất bại. Tôi sẽ tiếp tục đồng hành, tiếp tục chia sẻ những tâm huyết của mình, đặc biệt chú trọng các vấn đề về học thuật, về phương pháp và các bài học từ kinh nghiệm của bản thân. Bạn nào thấy có ích có thể đọc và theo dõi, thấy vô bổ chỉ cần bỏ qua là xong, đừng bận lòng mà chửi bới vì chả có giá trị gì.149Like

RRID:ZFIN_ZDB-GENO-190307-7

RRID:ZFIN_ZDB-GENO-191029-7

En somme, les études sur la communication des élèves atteints d’autisme permettent de mettre en évidence l’importance d’un contexte riche en stimulations appropriées (sons et images), mais également une évidente « stabilité » de l’information à décoder, le suivi des émotions des personnages, le rôle de l’imitation dans les apprentissages. Ces résultats encouragent donc l’usage d’outils informatiques adéquats pour améliorer la communication sociale chez les enfants atteints d’autisme.

Nous allons montrer par une courte analyse de quelques études l’impact du travail éducatif informatisé dans l’apprentissage de la communication sociale chez des enfants atteints d’autisme.

In the president's favor is the fact that his order commands the steel industry to follow policy set by Congress, not the president himself. The lawmaking power in the United States is within Congress's domain, but Congress has clearly chosen to rid itself of that power and give it to the president.

Looking at Johnson's actions, there is no evidence of an expressive element in his actions

El factor tiempo de trabajo está también muy relacionado con la variabilidad de la presión de inyección, ya que a medida que incrementa el tiempo de operación de un motor, se incrementa el desgaste de las piezas, siendo agravado el sistema de alimentación por la variación de la calidad del combustible

The Baltimore school district faced a crisis

7–2

5 percent of students used their vouchers at private schools.

7–2

dichotomous

Yet most companies neglect design as a strategy tool. What they don’t realize is that design can enhance products, environments, communi-cations, and corporate identity.”

maelstrom

dissenting

dissenting

ZFIN: ZDB-ALT-160901-7

ZDB-ALT-110309-7

RRID:ZFIN_ZDB-GENO-070702-7

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EducationSavings and investmentScientific researchHealthcareInternational tradeInfrastructureSpecial economic zones

Results for individual PALB2 variants were normalized relative to WT-PALB2 and the p.Tyr551ter (p.Y551X) truncating variant on a 1:5 scale with the fold change in GFP-positive cells for WT set at 5.0 and fold change GFP-positive cells for p.Y551X set at 1.0. The p.L24S (c.71T>C), p.L35P (c.104T>C), p.I944N (c.2831T>A), and p.L1070P (c.3209T>C) variants and all protein-truncating frame-shift and deletion variants tested were deficient in HDR activity, with normalized fold change <2.0 (approximately 40% activity) (Fig. 1a).

A total of 84 PALB2 patient-derived missense variants reported in ClinVar, COSMIC, and the PALB2 LOVD database were selected

SUPPLEMENTARY DATA

To this end, 44 missense variants found in breast cancer patients were identified in the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar) and/or selected by literature curation based on their frequency of description or amino acid substitution position in the protein (Supplemental Table S1).

Source Data

Source Data

Source Data

We, therefore, analyzed the effect of 48 PALB2 VUS (Fig. 2a, blue) and one synthetic missense variant (p.A1025R) (Fig. 2a, purple)29 on PALB2 function in HR.

Most Suspected Brugada Syndrome Variants Had (Partial) Loss of Function

we selected 73 previously unstudied variants: 63 suspected Brugada syndrome variants and 10 suspected benign variants

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Supplemental material

Supplemental material

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.

RRID:ZFIN_ZDB-GENO-190822-7

Full Card Number

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.

RRID:ZFIN_ZDB-GENO-200128-7

'[My] mouth [that] cursed you shall bless [you] as well!

Come, Shamhat, 1 will fix your destiny,

In [your presence] I will pray [to Anu,] father of the gods,

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.

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7

muscular hypotonia

intellectual disability

motor development

speech and language development

developmental delay

The only place he could think of was the Trianon in mid-town

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.

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

HE FUNERAL WAS HELD SEVERAL DAYS LATER AT THE BUDDHIST church up on the hill next to a playground.

They did not speak again until the car was beside the grocery store.

I went to Portland with him.”

‘Put my ashes in an orange crate and dump them in the Sound off Connecticut Street Dock where the sewer runs out,’

It scared him to think that he might be sobering up.

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.

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.

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.

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

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.

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.

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.

Accuracy (recovery

Iron Curtain

Romero was assassinated in 1980

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

Oral administration of doses

ROS assay

Cytotoxicity assay by Sulphorhodamine B (SRB) me

Cytotoxicity assay by MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5 diphenyltetrazolium Bromide)method

Cytotoxicityscreenin

Test for cardiac glycosides

Test for Terpenoids

Tests for Flavonoids

Tests for Phenolic compounds

Tests for Saponins

Tests for Tannins

Tests for Alkaloids

Test for Sterols

Qualitative study of phytochemicals of N. sativadurin

PCR amplification

Harvesting of cultures

Effect of pHon growth and sporulation of the bioagent

Effect of temperature on growth of bioagent

Effect of different media on growth of bioagentTrichoderma

Cultural characteristicsof bioagent Trichoderma

Relative distribution of virulence genes among V. parahaemolyticusfrom Cochin estuary, shrimp farm and sea food

Production of phosphatase

PCR amplification of 16S rRNA gene

DNA isolatio

Molecular confirmation of Vibrioby 16S rRNA gene se

Growth at different temperatures

Growth at different temperature

MAR indexingand antibiotic resistance pattern amon

Digestion of Plasmid DNA

Minipreparation of plasmid DNA from transformed co

Screening of recombinant E. coli clone

Transformation of ligation mixture in electro-competent E. coli host cells (DH5ααααstrain)

Transformation of ligated product in chemically competent E. coli host cells (DH5αααα strain)

Ligation reactions

Dephosphorylation of vector

Restriction digestion

Ligation of eluted PCR product in pBSK vector

Cloning of gel eluted PCR produc

Statistical analysis

PubChem Compound Database Screening

Assessmentof mitochondrial membrane potential(MMP or ΔΨm)

Growth kinetic studies

Kirby-Bauer Disk diffusion method

Antimicrobial activity

. Statistical analysis

Lactic dehydrogenase

Malic dehydrogenase

Succinic dehydrogenase

Estimation of dehydrogenases activities (SDH, MDHand LDH) in muscle and gill tissue of fish, Labeo rohita

Biological Yield(g)

Concentration of ideal carbon source

Post-vaccination declination of Transitional B cell population and increase in memory B cells in HBV positive newborns

Cell Counting

Semi dry transfer buffer

Stripping of Ni-NTA agarose beads

Cell transfection

GFP-β-cateninC-C (423-634 a.a) (ARM-C)

Chemicals/inhibitors

Sonication of bacteria

Cytotoxicity assay

ELISA dilution buffer

Lymphokines Assay

Gene cloning

Large scale plasmid extraction

Separation ofCurcuminoids

Estimation of Stevioside

Extraction from the plant material

Extraction from the plant material

Extraction from the plant material

Qmmtification of stevioside

Mesomorphic endomor~:

Humerus

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%

Purification oftlte semi-!)yntltetic a globin

Phlegyas

Quantitative screening for determination of xylanase in shake flask

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

Qualitative detection of xylanolytic activity by plate assay

DETECTION OF XYLANASE ACTIVITY

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.

ite directed mutagenesi

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.

Giemsa staining o/thin blood smear o/parasite cultures

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

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-

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)

Immunocytochemistry

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.

Deviations from ideal geometry of the various structures were analyzed using PROCHECK (R. A. Laskowski, 1993) from the CCP4 suite. PROCHECK

Validation, analysis and comparison of models

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.

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

Mini Plas~id Preparation and Screening

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

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.

Progesterone Radioimmunoassays

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.

Expression 'Of bZP3 in BEVS

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.

Sequencing of bZP3

minigel alongwith unligated vector to test the ligation. The ligated DNA was used to transform competent ~.coli cells.

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

Ligation of DNA fragments.

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.

Materials for autoradiography.

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.

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

Preparation of competent cells and transformation

Thecapabilityofheadkidneyneutrophilstomovewasassayedbyamigration-under-agarosetechniquemodifiedfromNelsonetal.(1975).ThemethodhasbeendescribedbySaloetal.(1998).Thedistance,thecellshadmigratedfromthemarginofthewelltowardsthewellcontainingcasein(directedmigration)andintheopposite direction(randommigration)weremeasuredunderthemicroscope.

Migration

Thereactionproduction,p-nitrophenolinacidphosphatewasmeasuredspectrophotometricallyat415nmagainstreagentblank.Theenzymeactivitywascalculatedfromthestandardcurveandexpressedasmicromolesofp-nitrophenolformedperhourpermilligramprotein.Therateofhydrolysisofp-nitrophenolphophateisproportionaltotheenzymepresentinthetissue.p-nitrophenylphosphate+NaoH—phosphat?-—>p -nitrophenol+phosphateThecolordevelopedinalkalinephosphataseactivitywasreadat410nmagainstreagentblankspectrophotometrically.Theactivityoftheenzymewasexpressedaspmolphenolformedmin'1mg'1protein

ThealkalinephosphataseactivitywasestimatedbythemethodofMorton(1955)usingp-nitrophenylphosphatesocolorlessinsolutionbutuponhydrolysis,thephosphategroupliberatesp-nitrophenylwhichishighlycoloredinalkalinesolution

Alkalinephosphatase(AKP:ortho-phosphoricmonoester-phosphohydrolase;E.C.3.1.3.1)

SamplesofC.punctataweretakenfromtheacclimationtanksandslightlyblottedonpapertowelstoremoveexcesswater.TheywereplacedindividuallyonaWhatmanfilterpaperinaonelitreglassbeaker.Theindividualswereimmediatelytransferredtobigglassdesiccatorscontaining250mlofthefollowingsolutionsfordesiredhumiditylevelsusinggradedsolutionsofKOHasdescribedbySolomon(1951).Watergiving95to97%relativehumidity(RH),mean95%;sodiumchloridegiving72to76%relativehumidity,mean75%;calciumchloridegiving28to31%relativehumidity,mean35%.Thedesiccatorswereplacedinanincubatorataconstanttemperatureof28°±1°Cwithdeterminationsofsurvivalandbodyweightatregularintervals.Thecontainerswereweighedatintervalstothenearest0.1mgandweightlosswasassumedtoequalwater loss

Survivaloutofwater