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Laboratory strain Ae. aegypti (Rockefeller) were maintained under constant environmental conditions (28°C with a 16:8 light:dark photoperiod). The mosquitoes were allowed to feed on bovine blood in Alsever’s anticoagulant via Hemotek feeding device (Discovery Workshops, Lancashire, England), after which the blood-fed females were sorted and allowed to digest the blood meal for 4days. Six cartons, each containing approximately 80 previously blood-fed females, were then intrathoracically-inoculated using an EntoSphynx Minucie (BioQuip, Rancho Dominguez, CA) dissecting needle dipped in viral stock. The control group of equal size (6 cartons of ~80 mosquitoes each) received an inoculation of media without virus.

After a 10-day extrinsic incubation period, mosquitoes were allowed to probe and feed on 1mL of 1× phosphate-buffered saline (PBS) and 10mM adenosine triphosphate (ATP) solution at 37°C in a Hemotek device for 1hour; a variation on the technique developed by Ribeiro, Rossignol, and Spielman[ 19 ]. This was repeated after 72hours to obtain a larger volume of PBS/ATP/saliva for downstream processing, approximately 12mL. After the collection of saliva, the inoculated cohort was taken down and their legs were removed and placed in 900μL of BA-1 diluent for viral RNA extraction and detection via qRT-PCR[ 26 ]. Disseminated infections were confirmed in >95% (n = 398/415) of the mosquitoes which supplied the ‘infected’ saliva solutions. From that dilute mixture, 200μL fractions of the PBS/ATP/saliva solution were added to 800μL of acetone chilled to −80°C and allowed to incubate at −20°C overnight. The tubes were then centrifuged at 4°C at 10,000rpm for 10minutes to pellet the precipitated protein. The supernatant was discarded and the resulting pellet was allowed to air-dry for 5minutes at room temperature. Once translucent, the pellet was then reconstituted in 100μL of 2-D rehydration buffer (Bio-Rad) consisting of: 8M urea, 2% CHAPS, 50mM dithiothreitol, 0.2% Bio-Lyte 3/10 ampholytes, and 0.001% bromophenol blue. The 100μL of reconstituted protein solution from the first tube was then used to reconstitute the second pellet, further concentrating the protein solution. This serial reconstitution was performed no more than 5 times to minimize loss of recovery from over-saturation. The concentrated protein solution still contained too much salt for 1 st dimension focusing; therefore the samples were processed through a ReadyPrep 2-D cleanup kit (Bio-Rad) and resuspended again in 2-D rehydration buffer. Protein concentrations were determined with a NanoDrop spectrophotometer (Thermo Scientific).

30μg of protein diluted in previously mentioned 2-D rehydration buffer to a final volume of 200μL was loaded per sample per 11cm, pH3–10 nonlinear IPG strip (Bio-Rad) overlaid with approximately 2mL of molecular biology-grade mineral oil (Bio-Rad) to prevent evaporation. First and second dimension electrophoresis conditions, gel staining, and imaging methods were performed as described previously[ Modern Tee SKYLINE I by VIDA VIDA Outlet Fashionable NQm3hhbO2

What is NIE?

Newspapers In Education (NIE) is an exciting interactive program that engages students in learning by providing teachers access to the Austin American-Statesman ePaper for use in their classroom. The NIE program is designed to make reading newspapers a fun and beneficial learning experience.

NIE is a worldwide effort in which school textbooks and teacher materials are supplemented with the use of the local newspaper. This is accomplished everyday by providing a “living textbook” in a format that is less intimidating and more appealing to students.

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This Week's lesson:

Lifetime job: Trump nominates Brett Kavanaugh as a Supreme Court justice

President Trump began this week with a major decision – his choice of Judge Brett Kavanaugh for a U.S. Supreme Court seat opening with this month's retirement of Justice Anthony Kennedy, 81, after 30 years on the nine-person court. The nominee, ...

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Are U.S. newsrooms under attack?

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NASA's Space Place

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This week's word in the news: CONTAINMENT



Determined by how much of the border of a wildfire has been cut off from sources of fuel.

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-- 07/09/2018

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