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black disciples founder
Black stands for all black people. When the program was active, there were 4 packages available for purchase, with each package granting more and more bonuses with increasing cost. Folks - ChicagoGangs.org Website. Les symboles du gang comprennent le chiffre romain trois ("III"), ce qui représente «l'esprit, le corps et l'âme», et est habituellement accompagné de l'abréviation BDN ("Black Disciple Nation"). It is also unique in its comparison with another African-American gang, the Gangster Disciples (GDs). The Black Disciples, sometimes abbreviated as BDN, BDN III, BD's, are one of the largest Chicago based street gangs. Adopted by: Folk Nation (Chicago), Gangster Disciples (Chicago), Black Disciples (Chicago) Why: "Hoyas," acting as an acronym for "Hoover's On Your Ass," pays homage to Gangster Disciples founder Larry Hoover. news 479 on now. Don Derky was also a co-founder of the Black Disciples and Barksdale's . By the beginning of 1961 David Barksdale, also known as "King David", took sole leadership of the Devils Disciples and appointed different members to oversee different areas of neighborhoods. [keywords: bloods and crips, bloods gang, piru bloods, blue bloods, bloods hand signs, crips and bloods, bloods knowledge, youngbloodz, bloods initiation, bloods 5 point star, bloods and crips, crips gang, crips signs, crips and bloods, stanley williams crips, hoover crips, tookie crips, sections: hiphop 650 on now. The gang has made several attempts to legitimize their image. wild'ish 277 on now. As of this writing, there may be as many as 300 "sets" of the BDs throughout the Chicagoland area. View the most prominent gangs based in Chicago in 2020. The race war between the Aryan Brotherhood and DC Blacks started on Nov 22, 1981 when the body of Robert M. Chappelle, a member of the DC Blacks was found dead in his cell at USP Marion.Thomas Silverstein (a high-ranking member of the Aryan Brotherhood) was the killer, and Chappelle's death worried bureau officials who thought it might spark a war, which it certainly did. Supermarket Shriek. ) Le gang compte environ 24 000 membres à travers Chicago et sa région (principalement), et le reste des États-Unis. Les BDS ont également une rivalité de longue date avec les Black P. Stones. Don Derky was also a co-founder of the Black Disciples and Barksdale's . Team 600 is a Chicago-based rap group who's members are also affiliated with the Black Disciples criminal gang.. Edai – Founder of Team 600. Nowhere in the New Testament does Jesus or Paul say he is rejecting Judaism and starting a new religion. Yummy Sandifer became a symbol of the gang problem in American inner cities, the failure of our countries social safety nets, and the shortcomings of the United States juvenile justice system. Team 600 is a Chicago-based rap group who's members are also affiliated with the Black Disciples criminal gang.. Edai – Founder of Team 600. Tragically, after committing murder, arson and armed robbery, he was murdered by his own fellow gang members who feared he could become an informant, and that he was attracting too much attention towards their activities. The Black Disciples are part of the alliance of street gangs but are known to engage in violence with other Folk Nation gangs such as the Gangster Disciples. King Von On Being The Black Disciples (BDN) founder David Barksdale's (King Dave) Grandson & More | NEW INTERVIEW; GRAPHIC FOOTAGE of FBG WOOSKI SHOT IN THE HEAD Surfaces + COMPLETE DETAILS x PICS!! There are three major bodies of the Disciples of Christ, all of which stem from a common source.. gear 23 on now. When we asked Edai on Twitter who the official members of Team 600 are, he replied "Great Question?.". Le gang fut créé à Chicago en 1976 par King Shorty. Entdecken Sie Veröffentlichungen von The Black Disciples auf Discogs. Multiply: Disciples Making Disciples [Download] Full Ebook. History's Number 1 Founder Chapter 555: Maturing disciples PREV ; NEXT A- A+ Chapter 555: Maturing disciples. Le gang est connu pour s'adonner au trafic de drogue, en plus d'autres activités criminelles. Players who purchased a package could also opt to upgrade to higher package tiers if they so desired. Don Derky was also a co-founder of the Black Disciples and Barksdale's . Les Black Disciples font partie de l'alliance Folks des gangs de rue mais sont surtout connus pour se livrer à des violences contre les autres gangs de Chicago comme les "Gangsters Disciples". Le gang fut créé à Chicago en 1974 par King Shorty. Members. Contrary to popular belief, #kingvon is NOT the grandson of Black Disciples founder, #davidbarksdale , aka #kingdavid . [ hide]. crips gang signs black stone-blood knowledge-pirus gang-tree top . By the late 1970's, in the wake of the assasination of the Nation's founder David Barksdale, it was decided that the Nation would no longer be a gang but an organization whose main focus was to make money. TBD Specialist Leader. David Barksdale (born Donise David Barksdale; May 24, 1947 – September 2, 1974), also known as King David, was an American gang leader from Chicago, Illinois.Barksdale was the founder of the Black Disciples.He and Larry Hoover decided to merge and create the Black Gangster Disciple Nation.Barksdale died on September 2, 1974, due to multiple gun shot wounds at age 27. Sep 20, 2009 . The Black Disciples (often abbreviated as BDN or BDN III) is a large black street gang based in Chicago, Illinois. Their founder was David "King David" Barksdale and they considered themselves a "Nation" (BGDN) not a gang. King Von is the Grandson of Black Disciples founder @hiphop. thots 227 on now. Black Disciples. The Black Disciples' symbol is the star of King David which has 6 points, and every point on the star has its own meaning; Love, Live, Loyalty, Unity, Knowledge, and Understanding. The Black Disciples are part of the alliance of street gangs but are known to engage in violence with other Folk Nation gangs such as the Gangster Disciples. tech 5 on now. Now free after 20 years in prison, he says he's trying to 'fix some of the stuff that we played a part in messing up.' King Von is the Grandson of Black Disciples founder @hiphop. zeta phi beta sorority founders poem in reply to 3 days ago . The President was David Barksdale and Vice President was Larry Hoover. The package tiers, bonuses, and costs are listed below; The following things listed below are exclusive to the Founders Program. CaptLeny. | VIEWER DISCRETION ADVISED!! The Black Disciples (BDs) are the Chicago "folks" gang that gained. As of this writing, there may be as many as 300 "sets" of the BDs throughout the Chicagoland area. Since they follow the original doctrine of David Barksdale they consider themselves the only true Disciples, as all other gangs have created their own literature and stra… Le gang fut créé à Chicago en 1976 par King Shorty. Blood Piru Knowledge : 09/20/09. When the leader and founder of the Black Gangster Disciple Nation, David Barksdale was slain, the gang adopted the symbol in his honor. news 479 on now. Matt Weed Co Underboss. In January 1969, David Barksdale, leader of eh Disciple Nation, developed a broader vision about the mission and purpose of street organizations (commonly referred to as "street gangs"). The Black Gangster Disciples began as the Devil's Disciples in the 1950s in Englewood. The Black Disciples (BDs) are the Chicago "folks" gang that gained international notoriety in 1994 by executing its eleven-year-old member "Yummy". When we asked Edai on Twitter who the official members of Team 600 are, he replied "Great Question?.". the Black Disciples, this can. He distributed drugs – including crack cocaine, powder cocaine, and heroin – and controlled the Black Disciples gang members' drug trafficking in the city of Chicago's far south side, including the violence-plagued Roseland and Altgeld Gardens communities. Vous pouvez partager vos connaissances en l'améliorant (comment ?) For that reason … Gangster Disciples est un gang qui a été fondé, dans les années 1960, du côté sud de Chicago , aux États-Unis, par Larry Hoover (en). Read: Profile of Rockford Gangster Disciples boss Karl Fort; 38-year-old Byron Montrail Purdy, known on the streets of Jackson, Tennessee, by his nicknames "Lil B" or "Ghetto," was the highest-ranking Gangster Disciple within the State of Tennessee, holding the title of governor. Dernière modification le 6 novembre 2019, à 19:35, Blue Fin Black Disciples Information and Pictures, Carte des territoires des gangs à Chicago, https://fr.wikipedia.org/w/index.php?title=Black_Disciples&oldid=164256106, licence Creative Commons attribution, partage dans les mêmes conditions, comment citer les auteurs et mentionner la licence. 600 (60th and King Drive) is a Black Disciple set. The Black Gangster Disciple Nation is a gang which was formed on the South-side of Chicago in the late 1960s, by David Barksdale, leader of the Black Disciples, and Larry Hoover, leader of the Supreme Gangsters. Cochise first connected with the Italian Outfit in 1971 and later became a co-founder of this organization, Boonie Black was another co-founder that was a hardened gangster that had no fear and was known to be serious business, it was even rumored that he shook down the Italian Outfit at one point in time. Contents. Barksdale's goal was to claim small gangs around the area and turn them into factions of the Disciples. The BDs also have a long standing rivalry with the Vice Lords, Black P. Stones, Black Souls & the Gangster Disciples (in Chicago and its suburbs). CHICAGO — A high-ranking leader of the Black Disciples street gang was sentenced today to 15 years in federal prison after being convicted of narcotics distribution. Les Black Disciples sont un gang de rue d'origine afro-américaine basé à Chicago dans l' Illinois. The Black Disciples were started in the year 1960 by David Barksdale. The Black Disciples are part of the alliance of street gangs but are known to engage in violence with other Folk Nation gangs such as the Gangster Disciples. Name Rank Left the guild Current guild Fame Exp R C Last seen Srv. There are other founding members who are called the original six which consists of … The Black Gangster Disciples aka the Gangster Disciples Nation is a gang that is under the Folk Nation. The two groups united to form the Black Gangster Disciple Nation (BGDN). As an Amazon Associate we earn from qualifying purchases. What are the official colors of The Black Disciples? By the mid-1960s, however, Barksdale became increasingly aware that the violence he and other gang member were regularly engaged during their … Soso Nord Black Gangster Disciple Nation - The Black Gangster Disciples began as . Edai responds on Twitter when asked "Who are the official members of Team 600 Ent?" Popular Songs From Edai Include: Cloude (cloude12) Ray Ray (rayray64) lee wilken (leewilken) Lists. Rico Recklezz ARRESTED!! thots 227 on now. Question Founder. GD founder Larry Hoover was born Nov. 30, 1950 in a small town in Mississippi. In 1966 to help increase recruitment and counteract threats of other gangs David Barksdale created the "Black Disciples Nation" which helped increased recruitment into the thousands. The Black Gangster Disciples are originally a Chicago gang. The Gangster Disciples is a dangerous gang founded in the 1970's in Chicago, Illinois. Its goal is to become a riddim section of the Disciple label. They were founded by D-Thang & Lil Boo. leewilken Owner. David Barksdale was the most important leader of the Devils' Disciples and after his death, Shorty Freeman would run the Black Disciples. King Von On Being The Black Disciples (BDN) founder David Barksdale's (King Dave) Grandson & More | NEW INTERVIEW CLICK BELOW To LINK w/ Rap Hitz!! The Gangster Disciples are a street gang which was formed in the South Side of Chicago in the late 1960s, by Larry Hoover, leader of the Supreme Gangsters, and David Barksdale, leader of the Black Disciples. movies 63 on now. The story begins on May 24, 1947 when David Barksdale was born in a small Mississippi town called Sallis.. We are always seeking new information regarding the Black Disciples, this can. See more ideas about gangster disciples, disciple, gangster. Surprisingly Barksdale survived and continued to run the Black Disciples. Le gang compte environ 24 000 membres à travers Chicago et sa région (principalement), et le reste des États-Unis. Xiao Yan, looking at Shi Tianhao, was simultaneously annoyed and amused. Hoover also organized the all-encompassing Folk Nation, which includes the Black Disciples, among other Chicago-area gangs. They have poor relations with other Folk NationGangs. Explorez les références de African Disciples sur Discogs. The defendant, WALTER BLACKMAN, 52, of Gary, Indiana, pleaded guilty in August 2014 to distribution of illegal narcotics. Disciples of Christ, group of Protestant churches that originated in the religious revival movements of the American frontier in the early 19th century. Nick Hussey Underboss. The colors of the Black Disciples are Black and Blue. movies 63 on now. In the mid-1980's, the groups splintered off from the BGD's all having their origins from the original Devil's Disciples street gang from the 1960's. It was discovered that Black Stone Rangers founder Eugene Hairston had ordered the shooting; he "commissioned" several 14-year-old children to kill Barksdale. The Black Disciples Nation (often abbreviated as BDs or BDN) is a Chicago 'Folks' gang that became notorious in 1994 due to their execution of an 11 year old member Robert "Yummy" Sandifer. Originally from Mississippi, selon les recommandations des projets correspondants. sports 383 on now. The 26-year-old man had been affiliated with O Block, a faction of Chicago's Black Disciples street gang, police say. Eac… Chicago gangs have been active in Atlanta for years. Their founder was David "King David" Barksdale and they considered themselves a "Nation" (BGDN) not a gang. Rev. Black Gangster Disciple Nation - The Black Gangster Disciples began as . Unfortunately, many famous Chicago rappers from Team 600 are members of the Black Disciples. rides 5 on now. sections: hiphop 650 on now. Le gang, toujours actif, possède une culture présentée comme une religion, une chaîne de commandement permettant une organisation structurée ainsi qu'un énorme réseau de trafic de drogues [ 1 ] . The organization has traditionally portrayed itself to the public as a legitimate yet aggressive agent of social and political reform. The Black Disciples is a large street gang based in Chicago, Illinois. The Black Gangster Disciples (GD) were formed in Chicago's Englewood neighborhood in the 1960's. The Black Disciples (often abbreviated as BDN or BDN III) is a large black street gang based in Chicago, Illinois. most propped. In Chicago, the Gangster Disciples have a long and bitter rivalry with the Black Disciples. The Goons in the Ickes joined the Black Gangster Disciple alliance with direct ties to David Barksdale and the Devil's Disciples. RAP HITZ ADS The Black Gangster Disciples are originally a Chicago gang. Larry Hoover, also known as "King Larry," is the notorious former leader of the Black Gangster Disciple Nation, a Chicago street gang that spread nationwide. Les Black Disciples sont un gang de rue d'origine afro-américaine basé à Chicago dans l'Illinois.Le gang est connu pour s'adonner au trafic de drogue et à la distribution, en plus d'autres activités criminelles. Larry Hoover had put together the Black Gangster Disciple Nation, which would vie for power and battle the Stones and Disciples for years to come. Edai responds on Twitter when asked "Who are the official members of Team 600 Ent?" Popular Songs From Edai Include: It is also unique in its comparison with another African-American gang, the Gangster Disciples (GDs). It was discovered that Black Stone Rangers founder Eugene Hairston had ordered the shooting; he "commissioned" several 14-year-old children to kill Barksdale. He moved with his family to Chicago in the late 1950s. Disciple Round Table was founded in September of 2017 by the recently signed artist 12th Planet. The Gangster Disciples are a street gang which was formed in the South Side of Chicago in the late 1960s, by Larry Hoover, leader of the Supreme Gangsters, and David Barksdale, leader of the Black Disciples.The two groups united to form the Black Gangster Disciple Nation (BGDN). 1 Background; 2 Intergang relations; 3 See also; 4 References . rides 5 on now. Blue is for the heavenly sky which blesses all Disciples. If you want to help support me expand into other cities and build my brand Please check out some of my merchandise below. In From Gangster Disciple to The Blueprint: Growth & Development, the BGD explain their history in their own words. sports 383 on now. Le gang est connu pour s'adonner au trafic de drogue, en plus d'autres activités criminelles. Kaufen Sie Platten, CDs und mehr von The Black Disciples auf dem Discogs-Marktplatz. TBD Head Caporegime. Unless otherwise stated, noneof these things will be re-released in the future. By the 1970's the Nation was split into two main factions, Black Disciples and Gangsta Disciples, which became known collectively as the Folk Nation. Category: Gang Gangs; Title: Black Disciple.. Coverage of Sandifer's death and retrospectives on his short, violent life were widely published in the American media. When the leader and founder of the Black Gangster Disciple Nation, David Barksdale was slain, the gang adopted the symbol in his honor. And about half of their 30 regional ministers are women — six of them Black women. We are always seeking new information regarding the Black Disciples, this can. The GD's and BD's became separate gang entities through splintering from the original Black Gangster Disciples. The Black Disciples (BD), also known as the Black Disciples Nation (BDN), are a large predominantly African-American street gang founded in 1960, by David Barksdale, on the South South of Chicago, Illinois. craigs list in waco 2 days ago . gear 23 on now. I don't think the Goons in ABLA were with the Disciples though since there really were no Disciples out there and that was the section Boonie directly oversaw. Jun 24, 2019 - Explore Demitris Mills's board "Gangster disciples" on Pinterest. The Churches of Christ emphasize rigorous adherence to the New Testament as the model for Christian faith, practice, and fellowship. The Black Gangster Disciples came from two different organizations that combined forces, the Black Disciples and the Gangster Disciples. David Barksdale; By the beginning of 1961 David Barksdale, also known as "King David", took sole leadership of the Devils Disciples and appointed different members to oversee different areas of neighborhoods.
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CIM Company
CIM 10 YEARS
Filmography and Awards
Artistic Residences
3,50 x 2,70 [three and a half two seventy]
CiM - Companhia de Dança / VOARTE
INTEREST AREAS *
The registration period for the 11th InShadow - Lisbon ScreenDance Festival, in the areas of video dance, documentary, animation, performance and visual arts, runs until June 30, 2019.
The applications will be submitted to a Jury formed by experts in the area of disciplinary crossings.
The awards for the Video-Dance and Documentary Competition are supported by Conserveira de Lisboa, ETIC - School of Technologies, Innovation and Creation, Adega Mayor and Associação Amigos da Cerca.
Inshadow'18 > Last Days
Inclusive Dance with Adam Benjamin > 10, 11, 12 Dec, Lisbon
Adam Benjamin is back in Lisbon in a preamble to the InArt - Community Arts Festival.
December 10, 11 and 12 at Ginásio Clube Português, the co-founder of Candoco Dance Company, accepted Voarte's invitation to share his experience and method in an intensive 3-day inclusive dance course. Adam has a fully inclusive understanding that involves the needs of each one. This course is intended for dance and theatre professionals, teachers and students. The subscriptions are open!
InShadow Festival'18 > Awarded Films
The first fifteen days of the InShadow Festival were marked by a strong adherence to the festival's initiatives. Yesterday at Teatro do Bairro were announced this year's winners in the areas of Video-Dance, Documentary and Animation. From 250 applications, were selected eighty two films for the international competition. The InShadow Jury - Documentary, Official, Schools, Audience, Vo'Arte and Children, had a demanding work awarding the 11 films in the various categories. Today Vo'Arte announces the winners of the 10th edition of the InShadow Festival 2018, in first hand. Congratulations to the winners and thanks to all the candidates and artists in the biggest edition ever!

redpajama_set_name: RedPajamaCommonCrawl


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Q: Copying content of one spreadsheet into another I have 2 different excel files, stored in C:\Test:

*

*Book1.xlsx, with Sheet1 (data in the Sheet1 is changing, not constant data-range, but always starts from A1 cell)

*Book2.xlsm, with Sheet2 (empty)

Every time after opening Book2, I need data from Sheet1 of Book1 to be automatically copied into Sheet2 of Book2.
Update:
I tried the following vba code (researched online from Excel Forum)
     Private Sub Workbook_Open()

     Application.EnableEvents = False
     Dim swb As Workbook, Lr As Long, LC As Long, sws As Worksheet
     Dim dCell As Range, srg As Range, dwb As Workbook, dws As Worksheet
     Set swb = Workbooks.Open("C:\Test\AAA\Book1.xlsx")
     Set sws = swb.Worksheets("Sheet1")
     Lr = sws.Cells(Rows.Count, 1).End(xlUp).Row
     LC = sws.Cells(1, Columns.Count).End(xlToLeft).Column
     Set srg = sws.Range(Cells(1, 1), Cells(Lr, LC))
     Set dwb = ThisWorkbook
     Set dws = dwb.Worksheets("Sheet2")
     Set dCell = dws.Range("A1")
     srg.Copy dCell
     swb.Close SaveChanges:=False
     dwb.Save
     Application.EnableEvents = True

    End Sub

But, the problem with this - that if I delete a few records after the 1st one from Sheet1 of Book1, then - these deleted records still appear in the Sheet2 of Book2!
I am not great in vba, it's just a part of my whole project
Sorry for these questions
Update 2:
When I have the following in Sheet1, Book1:

Then, after opening Sheet2, Book2, I see (which is correct, what I expected):

But, if I'll delete records in Sheet1, Book1 (starting from the 2nd):

Then, after opening Sheet2, Book2 I will still see those deleted from Book2 records (while I expect them to be gone):

A: When the destination sheet has more data than the source sheet, you will need to clear it before you copy over the data. You could do it with the Range-method ClearContents:
Set dws = dwb.Worksheets("Sheet2")
dws.Usedrange.ClearContents
Set dCell = dws.Range("A1")
srg.Copy dCell

As it seems that you are copying the data of the whole sheet, an alternative could be to remove the sheet in ThisWorkbook and copy the sheet itself:
' Delete Sheet2 (if present)
On Error Resume Next
Application.DisplayAlerts = False
ThisWorkbook.Sheets("Sheet2").Delete
Application.DisplayAlerts = True
On Error GoTo 0

' Copy Sheet from sourceWorkbook
sws.Copy after:=ThisWorkbook.Sheets(1)
ActiveSheet.Name = "Sheet2" ' Copied sheet gets the active sheet automatically.

A: Clear Range Below Copied Data
Private Sub Workbook_Open()

    Application.EnableEvents = False

    Dim swb As Workbook, sws As Worksheet, srg As Range, LR As Long, LC As Long
    Dim dwb As Workbook, dws As Worksheet, dCell As Range

    Set swb = Workbooks.Open("C:\Test\AAA\Book1.xlsx")
    Set sws = swb.Worksheets("Sheet1")
    LR = sws.Cells(sws.Rows.Count, 1).End(xlUp).Row
    LC = sws.Cells(1, sws.Columns.Count).End(xlToLeft).Column
    Set srg = sws.Range(sws.Cells(1, 1), sws.Cells(LR, LC))

    Set dwb = ThisWorkbook
    Set dws = dwb.Worksheets("Sheet2")
    Set dCell = dws.Range("A1")

    srg.Copy dCell

    With dCell.Resize(, srg.Columns.Count)
        .Resize(.Worksheet.Rows.Count - .Row - srg.Rows.Count + 1) _
            .Offset(srg.Rows.Count).Clear
    End With

    swb.Close SaveChanges:=False
    dwb.Save

    Application.EnableEvents = True

End Sub

redpajama_set_name: RedPajamaStackExchange


For sale very good townhouse with apartments.
It is located in Dobra Voda resort village at the distance of 200 meters from the beach.
The total area of this part of townhouse is 300 sqm.
The plot is 260 sqm.
The townhouse consist of 4 one bedroom apartments and one apartment with three bedrooms.
Excellent place for renting out.


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POST | This is Rochester
ART SCHOOL [article] || UofR VIDEO
An incredible partnership between the Memorial Art Gallery and the Rochester City School District gives kids access, opportunity, and inspiration.
BY JEN BRUNETT || PHOTOGRAPHS BY TOMAS FLINT
On particular Fridays from early fall to late spring, you may happen upon groups of children and adults weaving their way through the Neighborhood of the Arts, laughing, chatting amongst themselves, or generally enjoying their surroundings. You may pass them wondering what they're up to, smile and wave as you drive by, or simply dismiss them as part of your day. To the students, however, every step from School #23 on Barrington Street to the Memorial Art Gallery is a foot forward in learning, growing and making connections, whether they realize it or not.
The students of School #23 have a curriculum a bit different from that of their peers; instead of experiencing a six-and-a-half-hour school day, they have an extra hour. It's called Expanded Learning Time, and the purpose is to gain access to enrichment opportunities that they otherwise might not have access to. Within this supplementary time frame, students receive additional attention to their core curriculum, elective opportunities, and the ability to enhance their classroom learning by partnering with community organizations and businesses such as the Memorial Art Gallery, which will be starting their fifth year together in September 2017.
"We are an Expanded Learning Time school, which is a longer school day," says John Gonzalez, principal of School #23 and former director of Expanded Learning Time in the district.
"It's not after school, it's not before school, it's a long school day to do things differently for our students. We provide students with enrichment opportunities that they wouldn't necessarily get in a traditional school setting."
PHOTO CAPTION: Teacher Rose Van Tyne turns art concepts learned in the classroom into an understanding of the larger world.
Gonzalez says the reactions to the partnership from students and their families are very positive. "Parents volunteer to walk the mile with their children from school to the museum and form a bond in that way. Students are exposed to works of art that they potentially may not have been exposed to. Then they go into the art lab and create art based on what their learning was earlier in the day. They make interpretations of their own, ask questions, and form a deeper understanding."
The official title of the program is MAG RSCD ELT (Memorial Art Gallery Rochester City School District Expanded Learning Time) and was founded by Marlene Hamann-Whitmore, director of Academic Programs at the MAG, along with Rhonda Morien, then principal of School #23 and current principal of School #45. It has been cultivated since with love and compassion by a team of dedicated educators.
Sessions run for elementary students in ten-week intervals in the fall, winter, and spring on Fridays, which have expanded to Mondays to include schools #29 and #45. In the summer, program and school staff work together to create a new curriculum for the coming year, and they meet regularly to tweak the program according to the students' needs.
"Our job was to tie in as seamlessly as possible what the teachers were doing in the classroom to make strong curriculum connections, to help the children understand that what's going on in the classroom is not a discrete event," explains Whitmore. "It's one way of learning about that particular concept, which is really a larger part of the bigger world. Here's someone who's made a painting about what it's like to be in the natural world, or a sculpture as a reaction to war. Or a painting about the Erie Canal. They are actual living, breathing teaching documents, teaching with objects that relate to the concepts that are happening in the classroom."
Education for the young students goes beyond making classroom connections, as well. Marlene tells us that they work on 21st-century skills—non-cognitive, social and emotional skills such as holding doors for others, being respectful, using level zero voices, manners and respect.
Forward-thinking educators like Hamann-Whitmore and Gonzalez hope that others in the community will see how the students thrive in this type of environment and encourage them to use community resources and partner with supportive businesses.
Eight-year-old Colden Buzzell from School #23 has participated in the program during first and second grade. "I got to see lots of different kinds of art and then got to have a chance to try to do something similar," he says.
While at the MAG, he spotted a scroll with monkeys and a moon reflecting in the water. This led him to work on his favorite art piece, a scroll in which he drew a dragon-type creature, which resides now on his bedroom wall among other projects he has created. His mother, Leslie, says that he refers to the program as "super-duper."
This unique experience with the MAG also clicked unexpectedly outside of Rochester, when Leslie took her children to New York City.
"I was so surprised at his interest in the Metropolitan Museum of Art, where we spent a day. I thought we'd only get a couple hours in before the kids were bored, but because of his experience at the MAG (and my older daughter who was one of the first classes at #23 to do the program), they were very comfortable in the art museum setting, looked at the art, read the signs, and had things to say about the pieces we looked at. I credit their MAG experience with that!"
If you happen to pass a group of children in the Neighborhood of the Arts walking with a sense of purpose, give them a respectful wave or a smile knowing that you're helping to make a friendly connection from classroom, to their environment, to the arts and, finally, expression.
POST | ISSUE 23 FALL 2017
Watch a UofR produced video on the MAG–RCSD Expanded Learning Collaboration


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Europe's air safety authority declares Boeing's 737 MAX safe to return to the skies, after deadly crashes grounded it for 2 years
Grounded Boeing 737 MAX aircraft are seen parked (FILE PHOTO) © REUTERS/Lindsey Wasson
The European Union Aviation Safety Agency (EASA) has given the green light for a modified version of the Boeing 737 MAX to return to service, but issued a package of measures that must be completed before aircraft can fly.
On Wednesday, the EASA gave its approval for the Boeing 737 MAX to return to the skies in Europe after two years on the ground.
The aviation body said the aircraft had met the four conditions for a return to service that it had laid down, but mandated a number of required modifications and upgrades which airlines must undertake if the plane is to take to the skies. These include software upgrades, electrical reworking, maintenance checks, operations manual updates and crew training.
"We have reached a significant milestone on a long road," said EASA Executive Director Patrick Ky, noting, "we asked difficult questions until we got answers and pushed for solutions which satisfied our exacting safety requirements."
"We have every confidence that the aircraft is safe, which is the precondition for giving our approval. But we will continue to monitor 737 MAX operations closely as the aircraft resumes service," Ky added.
Also on rt.com EU to give flight clearance to Boeing 737 MAX next week following similar moves in US and Brazil
The aircraft was universally banned from taking to the skies after two deadly accidents in late 2018 and early 2019 but began flying again in the US, Brazil, Panama and Mexico in late 2020.
The two accidents claimed 346 lives. In October 2018, Lion Air Flight 610 crashed into the Java Sea 13 minutes after takeoff, killing 189 people. In March 2019, Ethiopian Airlines Flight 302 crashed near the town of Bishoftu just six minutes after takeoff, killing all 157 people on board.
The EASA noted that the root cause of these tragic accidents was traced to software known as the MCAS (Maneuvering Characteristics Augmentation System). The software was intended to make the plane easier to handle.
Trends:Boeing newsEurope news
'Weeping' over 'brave corporations' like Amazon, Google & Apple? You might have Stockholm Syndrome… or just work for Buzzfeed
Billionaire brawl: Musk blasts Bezos' Amazon over effort to 'hamstring' SpaceX Starlink satellite internet project


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The Help Group Launches its New Paws and Pals Program
The Help Group is pleased to announce the launch of Paws and Pals for Kids with Autism, a volunteer-supported pet intervention program offered at its Sherman Oaks and Culver City Campuses. Volunteers from Therapy Dogs, Inc. and Autism Service Dogs of America visit The Help Group's autism schools on a weekly basis with their specially trained and registered therapy and service dogs.
Students attending The Help Group autism schools and camps last summer were the first to be greeted by volunteer Vicky Marsh and her dachshund Clyde. Volunteers like Vicky are placed in a variety of small group settings, including classrooms and day camps, where they have the opportunity to facilitate interaction between their pets and the students. Vicky shared, "It was such a rewarding experience spending time with the amazing children and staff. The kids are happy and enthusiastic and greeted Clyde and me with warm smiles and happy hearts. The Help Group is a special place."
"Animals can have a very positive impact and present an avenue for social interaction and communication," said Dr. Barbara Firestone. "The Help Group is thrilled to offer this dynamic new program."
Students show increased motivation in the presence of handler-and-dog teams, which can be a valuable tool for teachers to help students initiate conversation; share in reciprocal exchange, and social interaction with peers. Students from Village Glen School practiced reading and letter writing using the visiting dogs as the topic. Non-verbal students in Sunrise School are using their Picture Exchange Communication System (PECS) to communicate about the dogs in their classrooms.
Although some children may find it difficult to relate to and communicate with their peers, relating to animals can be a natural, soothing and non-judgmental experience. "Handler-and-dog teams can encourage social interaction and boost our students' self-esteem and confidence," said Pamela Clark, Director of The Help Group Autism Schools. "Pet intervention programs can also lead to increased motivation and decreased anxiety in our students."
Paws and Pals teams have already integrated into classrooms to achieve these educational objectives and to help students develop skills that can ultimately be transferred to relationships with peers and teachers.
A very special thanks to the remarkable Paws and Pals volunteers for sharing their pets with our students and to Lisa Manafian, Help Group Special Projects Coordinator, for her efforts in developing this program. Paws and Pals is off to a great start!
Note: This article appears in the Winter 2013 edition of HelpLine. Follow this link to download the newsletter.
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Finally, Law On Property Sharing Between Boyfriend And Girlfriend Upon Separation In Final Stages
The law on property sharing within cohabiting relationships (boyfriend/girlfriend) is in its finals stages, awaiting tabling on the floor of Parliament for legislation.
Spearheaded by the Uganda Law Reform Commission (ULRC), the proposed legislation seeks to draw modalities on how property acquired among couples that aren't legally married, but have been living together, will be shared upon demise of the relationship.
Led by Vastina Rukimirana (Chairperson ULRC) the officials of the Commission had appeared before the Legal and Parliamentary Affairs Committee to present their 2018/2019 Ministerial policy statement.
Among the activities to be undertaken in the coming year is the development of legislation to govern sharing of property in cohabitation relationships.
Rukimirana told the Committee that following consultations undertaken on the Marriage and Divorce Bill 2009, the Commission was advised to strike out the controversial clauses, if the Bill was to see the light of the day.
It should be recalled that the matter on property sharing was a subject of contention during the consideration of the Marriage and Divorce Bill 2009, but the clause on property sharing received a cold shoulder from MPs.
Clause 127(1) of the Bill states that where a spouse acquires property before or during the marriage and the property does not fall within matrimonial property as defined in section 115, but his or her spouse makes a contribution towards the improvement of that property, be it monetary or in kind, the spouse shall acquire a beneficial interest equivalent to the contribution she or he made.
Still clause 141(1) notes that upon separation of the spouses, matrimonial property shall not be divided between the parties but a court may order that the spouses share any income that may accrue from the matrimonial property.
The other point of contention was marital rape that received fierce reception from the male MPs in the 9thParliament. This prompted Parliament to facilitate each MP with Shs5M to consult their voters on the matter.
During the Committee session, Oboth Oboth MP (West Budama County) narrated his ordeal during the consultations on the Marriage and Divorce Bill in his constituency.
He said the locals rejected the proposal in the Bill to outlaw marital rape saying the electorates argued that in the japadhola culture, they know they can never get anything (sex) on a silver platter and warned Parliament against 'Americanising' their culture.
The Commission revealed that following the protest against the Bill, there was an agreed position to strike out the contentious clauses and rename the bill; Marriage Bill.
Wilfred Niwagaba, the Shadow Attorney General welcomed the development saying tabling the Bill of such nature is long overdue explaining that couples in cohabiting relationships (who are the majority in Uganda) need this Bill the most.
"The law on sharing of property is long overdue if we actually kill the phobia around the Marriage and Divorce Bill. Most women are used and abused and at the end, they walk out empty handed," Niwagaba said.
In their budget proposal, the Commission is seeking for Shs6.5Bn that would be used for printing the 7thedition of the Laws of Uganda, both principal and subsidiary laws.
However, the money hasn't been provided for in the upcoming budget despite the fact that the Commission is lagging behind by three years to undertake this exercise.
In the 2018/2019 budget, the Commission budget proposal is Shs18.58Bn, of which Shs5.397Bn (wage), while Shs12.823Bn for non-wage recurrent expenditure and Shs366.450Bn for development expenditure, with the above allocation including Shs6.5Bn for printing the revised editions and Shs800M to translate and print translated constitution in Luganda, Leb-Acholi and Kiswahili.
However, the Ministry of Finance has only provided Shs17,262,947,000Bn.
The Commission also intends to translated the Constitution into Leb-Lango and Rufumbira with the Commission informing the Committees that the Attorney General has been threatened with law suits, if the constitution isn't translated in other languages as required by law.
By Stella Mugoya
Related Topics:Home
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Two of Uganda's Very Important Persons have Monday died.
National Unity Platform's parliamentary flag bearer in the last year's Omoro County by-elections, Simon Toolit Aketcha is dead.
Toolit, died on Monday morning.
The news of his death was confirmed by Omoro county MP Andrew Ojok Oulanyah.
"It is a dark day in Omoro! We have lost one of our gallant sons and former MP, Hon. Simon Toolit Akecha! Hon. Toolit was the MP for Omoro County between 2006 & 2011 and was a candidate in recent by-elections. May his soul rest in eternal peace!" OJok tweeted.
Toolit put up stiff political resistance against the late Jacob Oulanyah before also competing against his son Andrew Ojok Oulanyah.
Toolit will be remembered as a candidate to have competed against the Oulanyah's while holding the flag for two different parties for under ten years.
In 2016, Toolit, then a candidate of Forum for Democratic Change (FDC) contested against NRM's Jacob Oulanyah and polled 6,823 votes against the latter's 8,218 votes.
In the same manner, Uganda's high commissioner to Kenya and Seychelles, Dr Hassan Galiwango, has died.
Galiwango died Monday morning at Nairobi Hospital where he had been admitted.
The late was the husband to Mbale City Woman Member of Parliament, Connie Nakayeze Galiwango.
President Museveni appointed Galiwango as the high commissioner to Kenya in October 2020.
Galiwango was also the former long serving director of finance and administration at the NRM secretariat at the time of his ambassadorial appointment. Ms Medina Naham, the NRM party chairperson of Koboko District, replaced him at the secretariat.
The cause of his death is yet to be communicated by the government.
Last year, Bank of Uganda governor prof. Emmanuel Tumusiime-Mutebile, Speaker of Parliament Jacob Oulanyah and Gen. Elly Tumwine among other VIPs died.
A bomb killed at least five people on Sunday after detonating in a church in eastern Democratic Republic of Congo, the army said.
Details of the attack remain hazy, but military spokesman Antony Mualushayi said the "terrorist act" happened in a Pentecostal church in North Kivu province's Kasindi, a town on the border with Uganda.
Mualushayi later told reporters that at least five bodies had been taken to the morgue following the blast, in what he called a provisional death toll.
Joel Kitausa, a local civil society figure, put the death toll at eight and said 27 other people had been wounded.
AFP was unable to independently confirm the toll.
It was not immediately clear who carried out the attack. Mualushayi said that one suspect — a Kenyan — had been arrested.
The DRC's communications ministry said on social media that the attack was apparently carried out by the Allied Democratic Forces (ADF) group.
The ADF — which the Islamic State group claims as its affiliate in Central Africa — is active in the Kasindi area.
It has been accused of slaughtering thousands of Congolese civilians and carrying out bomb attacks in Uganda.
The ADF has planted bombs in towns in North Kivu in the past.
Since 2021 a joint Congolese-Ugandan military operation has been targeting the ADF in North Kivu and neighbouring Ituri province.
Over 120 armed groups roam mineral-rich eastern DRC, many of which are the legacy of regional wars that flared at the turn of the 21st century.
Dozens of people have been killed after a plane with 72 people on board crashed near an airport in central Nepal.
The Yeti Airlines flight from Kathmandu to the tourist town of Pokhara crashed on landing before catching fire.
Videos posted on social media show an aircraft flying low over a populated area before banking sharply.
At least 68 people are confirmed to have died, officials said. Several critically injured survivors were taken to hospital, unconfirmed reports said.
Local resident Deeveta Kal told the BBC how she rushed to the crash site after seeing the aircraft plunge from the sky shortly after 11:00am local time (05:15 GMT).
"By the time I was there the crash site was already crowded. There was huge smoke coming from the flames of the plane. And then helicopters came over in no time," she said.
"The pilot tried his best to not hit civilisation or any home," Deevta Kal added. "There was a small space right beside the Seti River and the flight hit the ground in that small space."
The flight set out with 68 passengers on board, including at least 15 foreign nationals, and four crew members.
According to flight tracking website Flightradar24, the 15-year-old twin-engine ATR 72 stopped transmitting position data at 05:05 GMT and the last signal from the aircraft was received at 05:12.
Hundreds of Nepalese soldiers were involved in the operation at the crash site in the gorge of the Seti, just one and a half kilometres from the airport.
The search operation has been suspended for the day, officials say.
Video taken where the plane came down showed thick billowing black smoke and burning debris.
"We expect to recover more bodies," an army spokesman told Reuters, saying the plane "has broken into pieces".
Prime Minister Pushpa Kamal Dahal called an emergency meeting of his cabinet and urged state agencies to work on rescue operations. A panel to investigate the cause of the crash has been set up.
Of the passengers, 53 are said to be Nepalese. There were five Indian, four Russians and two Koreans on the plane. There was also one passenger each from Ireland, Australia, Argentina and France among others.
Aviation accidents are not uncommon in Nepal, often due to its remote runways and sudden weather changes that can make for hazardous conditions.
A Tara Air plane crashed in May 2022 in the northern Nepalese district of Mustang, killing 22 people.
In early 2018, 51 people were killed when a US-Bangla flight travelling from Dhaka in Bangladesh caught fire as it landed in Kathmandu.
The European Union has banned Nepalese airlines from its airspace over concerns about training and maintenance standards in the country's aviation industry.


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Speech and Debate Team Continue on Winning Streak
Jennifer Tinoco
The Speech and Debate team was victorious in a California State competition this past weekend in Woodland Hills, earning four gold medals, three silver medals, two bronze medals, and placing sixth out of the 28 community colleges that participated.
"The State competition is open to all community colleges in California, and there is no qualification for it," said Ira Heffler who is a Language Arts instructor on campus. "We are really confident this year."
The State competition took place at the Marriott hotel.
"Since it's such a huge competition, we wouldn't be able to fit in a school," said the Language Arts Division Chair Jean Perry. "It gets so crowded with students from all over California and we have various rounds that the judging differs from that of our other competitions throughout the year," added Perry.
"During a competition, when someone stands up to speak, the audience consists of other students and faculty members from other schools.
"The judges who are present turn in their ballots after a presentation is done. Those ballots are then taken to the judging room.
"We also have what is called the 'elimination round' and that is when the students who have tied in previous rounds go head to head," she added.
The Speech and Debate club is also known as the Forensics Team because just like the crime scene investigators, the club members must also practice and do a lot of research to back up their debates during competitions.
When it comes to competitions, some people may think about the nerve-wracking feelings that emerge before one speaks in front of a large audience. Although Robert Cannon said it is not like that at all.
"It is not really about nervousness anymore," said Cannon, who is the President of the Speech and Debate Club. "Honestly it was a very tiring competition, but it served as a warm up for our Nationals coming up in April," added Cannon, who also won a gold medal for poetry interpretation.
The state competition lasted four entire days "in which a lot of our energy was consumed, but it was worth it," said Cannon.
"This year our national tournament will be in the University of Minnesota where we will be competing against four-year universities as well," said Perry.
"Just last year at nationals, our team ranked at third place," said Heffler. "And we hope that we can beat that number this year."
The National Competition for Minnesota is for both two-year and four-year colleges and universities. Meanwhile there is a separate national competition for two-year colleges only that takes place in Texas.
"In order to compete at the Minnesota Nationals we must have members of the team who qualify, and so far we have four that are ready to go," said Perry.
"We are the only two-year college that is competing in both Nationals this year and I think we have a great shot at it," said Cannon.
The Forensics team will also hold a two day Speak – O – Fest on campus starting April 24 and April 25 at the Student Center from 11 a.m. to 1 p.m. Free food and free entertainment will be provided as well.
"We have a variety of speeches and interpretations on both days, said Cannon. "We have some pretty fun stuff to share with the campus."
"This [ Speak- O – Fest] is also a great time for students to learn what our team is all about and if anybody is interested in joining, our festival will certainly be a great time to consider it and you'll get absolutely hooked," said Heffler.
The forensics team meets in AD 205 every Thursday from noon to 1 p.m. Perry may be reached at (818) 240-1000 ext. 5504, and Heffler may be reached at ext. 3113.


Paul Scarlato has represented the interests of investors since 1989. Among his more recent efforts are the Deutsche Mortgage Pass-Through Securities Litigation ($32.5 million settlement for purchasers of mortgage backed securities ); Lehman Brothers Equity/Debt Securities Litigation (settlements over $500 million for Lehman debt and equity purchasers); Velti plc Securities Litigation (partial settlement of $9.5 million for Velti stock purchasers); Hewlett Packard Securities Litigation ($57 million settlement for HP common stock purchasers); In Compellent Technologies, Inc. Shareholder Litigation (settlement in connection with proposed acquisition of Compellent Dell, Inc. included key deal improvements; elimination of "poison pill" and standstill agreement with potential future bidders, and a reduction of termination fee); and In re Boeing Derivative Litigation (following a series of government scandals involving Boeing; settlement for shareholders included allocation of $29 million to establish and innovate ethics and compliance programs, and required the board to examine company's entire governance structure). Paul has an undergraduate degree in accounting and was part of the tax department of a "Big Six" accounting firm where he provided a broad range of services to large business clients in a variety of industries prior to entering the securities litigation practice.
Following his judicial clerkships, Mr. Scarlato became a member of the tax department of a "Big Six" accounting firm where he provided a broad range of services to large business clients many industries.


There are no approved small molecule drug therapies for human respiratory syncytial virus (hRSV), a cause of morbidity and mortality in at-risk newborns, the immunocompromised, and the elderly. We have investigated as a potential novel hRSV drug target the protein-protein interaction between the C-terminus of the viral phosphoprotein (P) and the viral nucleocapsid protein (N), components of the ribonucleoprotein complex that contains, replicates, and transcribes the viral RNA genome. Earlier work by others established that the 9 C-terminal residues of P are necessary and sufficient for binding to N.
We used a fluorescence anisotropy assay, surface plasmon resonance and 2-D NMR to quantify the affinities of peptides based on the C terminus of P for RNA-free, monomeric N-terminal-truncated N(13-391). We calculated the contributions to the free energies of binding of P to N(13-391) attributable to the C-terminal 11 residues, phosphorylation of the C-terminal 2 serine residues, the C-terminal Asp-Phe, and the phenyl ring of the C-terminal Phe.
Binding studies confirmed the crucial role of the phosphorylated C-terminal peptide D(pS)DNDL(pS)LEDF for binding of P to RNA-free, monomeric N(13-391), contributing over 90% of the binding free energy at low ionic strength. The phenyl ring of the C-terminal Phe residue contributed an estimated -2.7 kcal/mole of the free energy of binding, the C-terminal Asp-Phe residues contributed -3.8 kcal/mole, the sequence DSDNDLSLE contributed -3.1 kcal/mole, and phosphorylation of the 2 Ser residues contributed -1.8 kcal/mole. Due to the high negative charge of the C-terminal peptide, the affinity of the P C-terminus for N(13-391) decreased as the ionic strength increased.
The results support the idea that the interaction of the C-terminal residues of P with N constitutes a protein-protein interaction hotspot that may be a suitable target for small-molecule drugs that inhibit viral genome replication and transcription.
Human respiratory syncytial virus (hRSV) is a non-segmented negative sense RNA virus in the order Mononegavirales, the family Paramyxoviridae and the genus Pneumovirus, and is the cause of severe respiratory tract infections among newborns, immunocompromised people, and the elderly . Current treatments are limited to antibody prophylaxis with palivizumab for high-risk newborns, general antiviral therapy with ribavirin, and supportive care. Efforts to develop anti-RSV vaccines and new drugs to prevent and treat the disease have so far been unsuccessful -.
The genome of hRSV codes for 11 proteins. Four of these, the polymerase (L), nucleocapsid protein (N), phosphoprotein (P) and transcription anti-termination factor (M2-1), along with the RNA genome, make up the ribonucleoprotein (RNP) complex responsible for transcription and replication of the RNA. Several types of low molecular weight compounds have been identified that appear to target the RNP. Mutations conferring resistance to the inhibitory effect of YM-53404 and related compounds on viral replication were found in the polymerase L ,. Mutations conferring resistance to RSV604 are found in the nucleocapsid N protein . Liuzzi et al.  identified inhibitors of the mRNA capping function of the L protein, resistance mutations to which are found in the L protein. Thus the RNP is a viable target for compounds that inhibit RSV replication.
Several protein-protein interactions are involved in assembly of RNP. The N protein forms helical oligomers around which the RNA is wrapped ,. P protein oligomerizes , and interacts with N -, M2-1-, and L -. M2-1 is a tetramer , and interacts with N - as well as P. These protein-protein interactions may offer suitable targets for antiviral drug discovery.
Protein-protein interactions can be difficult to disrupt with low molecular weight drugs if the interactions occur over a large, flat surface. Protein-protein interaction hotspots, confined areas with deep topological features, however, offer greater potential for drug discovery . The information available concerning the interaction of the C-terminus of the RSV P protein with the N protein suggests that this interaction may occur at such a hotspot. A putative P binding region on the N-terminal core domain of N was identified by Galloux et al. , including Lys 46, Met 50, Ile 53, Ser 131, Arg 132, Tyr 135, Arg 150, and His 151. Mutations of several residues in this domain to alanine reduced both viral RNA synthesis and the N-P interaction in vivo and in vitro. On the crystal structure of N, these residues are clustered together, forming a potential P binding site with a hydrophobic cavity surrounded by several basic side chains. Remarkably, only the C-terminal 9 residues of P (Asp 233 to Phe 241) are necessary for binding to N in pull-down assays ,. Moreover, changing just the C-terminal phenylalanine residue of P to alanine abrogates both N binding and viral RNA synthesis . These observations strongly support the existence of a P-N binding hotspot involving the C-terminus of P and the aforementioned N-terminal core domain of N.
The C-terminal 11 residues of P (Asp-Ser-Asp-Asn-Asp-Leu-Ser-Leu-Glu-Asp-Phe) include 2 serine residues, Ser 232 and Ser 237, both of which have been observed to be phosphorylated, although the importance of their phosphorylation is unclear. Mazumder et al.  showed that purified recombinant P was selectively phosphorylated on Ser 237 by casein kinase II. Mazumder and Barik  found that mutation of Ser 237 to Ala abrogated RNA transcription by reconstituted RNP in vitro, whereas mutation of Ser 232 to Ala had no effect. In contrast, Barik et al.  found that Ser 232 was the major site of P phosphorylation when it was expressed in human cells or when unphosphorylated recombinant P was treated with a crude human cell extract. Sánchez-Seco et al.  found that Ser 232, not Ser 237, was the main site of phosphorylation of recombinant P in vitro by casein kinase II. Villanueva et al.  found that phosphorylation of P expressed in human cells was mainly on Ser 232 and to a lesser extent on Ser 237, based on the decreased level of phosphorylation when either residue was changed to Ala, but that mutation of either of these residues had little effect on viral transcription or replication. Only mutation of both residues combined with mutation of other probable sites of serine phosphorylation elsewhere in the protein substantially reduced, but did not completely eliminate, transcription and replication. Finally, Lu et al.  found that eliminating phosphorylation of Ser 232 and Ser 237 by mutation reduced P phosphorylation by 80% in infected cells, but had only modest effects on reporter gene expression and the N-P interaction. The mutant virus showed impaired replication in rodents compared with the wild-type virus, however. The inconsistencies in the results reported in the literature on this topic do not allow us to draw definitive conclusions regarding either the extent of phosphorylation of Ser 232 and Ser 237 or the roles of these residues in the functions of P, including its interaction with N.
We have taken a quantitative biophysical approach to investigate the P-N protein-protein interaction with regard to the effect of phosphorylation of the C-terminal N-binding peptide of P on residues 232 and 237 and the importance of the C-terminal Phe residue. In the process, we demonstrate 3 practical measurement systems – fluorescence anisotropy, surface plasmon resonance, and 2-D NMR - that can be used as the first steps in drug discovery programs to identify inhibitors of the interaction.
Peptides having the sequence of the C-terminal 11 residues of RSV P protein (DSDNDLSLEDF) were labeled on the N-terminus with the fluorophore BODIPY FL for fluorescence anisotropy-based measurements of binding to N(13-391), an N-terminally truncated construct of RSV N protein that does not oligomerize . The truncation makes the protein easier to overexpress in soluble form in bacteria than full-length N. When purified from bacteria, the truncated N proteins used in this study had no detectable RNA associated with them. One peptide was unphosphorylated (BP1). The other was phosphorylated on both serine residues (BP5), corresponding to Ser 232 and Ser 237 of the P protein. Both peptides bound to the N(13-391) construct (Figure 1A). The affinity of the doubly phosphorylated peptide BP5 (Kd = 78 ± 5 nM) was about 10 times that of the unphosphorylated peptide BP1 (Kd = 760 ± 20 nM). This result indicates that phosphorylation of the serine residues in the C-terminus of P is likely to affect the affinity of the N-P binding interaction. The anisotropy change was also larger for BP5 than BP1. Subsequent experiments were performed with BP5 because of the lower N(13-391) concentration required to perform competition experiments and larger signal compared with BP1.
Fluorescence anisotropy measurements of P C-terminal peptide binding to N(13-391), effect of KCl, and competition by full-length P. (A) Affinity of unphosphorylated and doubly phosphorylated P-derived C-terminal peptides BP1 and BP5, respectively, for N(13-391). Data points and error bars represent the means and standard deviations, respectively, of 3 replicates. The error bars are mostly hidden by the data points. The Kds for BP1 and BP5 were 760 ± 20 nM and 78 ± 5 nM, respectively (best-fit value ± standard error of fit). (B) Effect of KCl concentration on affinity of BP5 for N(13-391). Kds were measured with N(13-391) concentrations ranging from 1.95 to 2000 nM in triplicate. The data were fit as above. Data points and error bars represent the best-fit Kds and the standard errors of the fits, respectively. (C) Inhibition of BP5 binding to N(13-391) by full-length P protein. The IC50 was 180 ± 10 nM (best-fit value ± standard error of fit).
Considering the highly negative charge of the doubly phosphorylated P C-terminal peptide due to the presence of 2 phosphate groups and 5 acidic side chains within the 11 residue peptide, we reasoned that electrostatic interactions with N would likely play a significant role in the interaction between them. Moreover, the putative P binding site on N contains several basic amino acid side chains . In support of this hypothesis, we found that the affinity of BP5 for N(13-391) diminished as the ionic strength increased (Figure 1B). Subsequent experiments were performed in a low-ionic strength buffer in order to maximize the affinity of BP5 for N(13-391), thereby minimizing the amount of protein required.
The effect of phosphorylation of each of the two serine residues in the C-terminal peptide on its affinity for N(13-391) was investigated by competition with BP5 binding (Table 1). Phosphorylation of either serine residue substantially increased the affinity of the peptide compared with the unphosphorylated peptide, 7-fold for the N-terminal serine and 10-fold for the C-terminal serine. Phosphorylation of both serine residues resulted in an additional affinity enhancement of approximately 3-fold greater than observed for phosphorylation of one residue. As a result, the affinity of the doubly phosphorylated peptide was 20-25-fold higher than the affinity of the unphosphorylated peptide. This is consistent with the above observation that the affinity of doubly phosphorylated BP5 is 10-fold higher than the affinity of unphosphorylated BP1.
Competition was measured by the fluorescence anisotropy assay and with N(13-391) binding to P by SPR. Competitor peptide concentrations ranged from 0.2 to 200 μM. The IC50 for D(pS)DNDL(pS)LEDF was measured twice using fluorescence anisotropy. IC50s from the fluorescence anisotropy assay are approximately twice the Kds of the competitors because the N(13-391) concentration was set at the Kd for the interaction of N(13-391) with BP5. ND, not done.
The Kds of the peptides in the fluorescence anisotropy experiments are approximately 1/2: the IC50s because the N(13-391) concentration used was equal to its Kd with BP5. Therefore, the Kds of DSDNDLSLEDF and D(pS)DNDL(pS)LEDF were about 7.6 and 0.34 μM, respectively. These Kds are considerably higher than the Kds of the same peptides bearing an N-terminal BODIPY FL fluorophore, BP1 (0.76 μM) and BP5 (0.078 μM), respectively. This is most likely due to the hydrophobic character of the fluorophore, which tends to enhance the affinity of a ligand as long as the fluorophore does not cause a steric clash.
The C-terminal phenylalanine residue of P is crucial for binding of P to N, and mutation to alanine abrogates binding and viral replication . The effect of changing the C-terminal Phe residue to Ala on competition by the P C-terminal peptide with BP5 binding to N(13-391) was substantial (Table 1). For unphosphorylated peptides, the IC50 increased by more than 13-fold when Phe was changed to Ala. The IC50 of the Ala-modified peptide exceeded the highest concentration tested (200 μM). Inhibition by 200 μM peptide was about 25%, so the true IC50 can be estimated to have been approximately 600 μM, assuming a Hill slope of 1, which was typical for these measurements. Thus the IC50 shifted upward about 40-fold when Phe was replaced with Ala in the unphosphorylated peptide. For the phosphorylated peptides, changing Phe to Ala resulted in an upward shift of the IC50 by approximately 100-fold. These results are consistent with the literature showing that the C-terminal Phe of P is a crucial binding determinant for its interaction with N.
Full-length P protein produced in insect cells was tested for its ability to compete with BP5 binding to N(13-391). As determined by mass spectrometry, the P protein consists of a mixture of unphosphorylated, singly and doubly phosphorylated species, with the singly phosphorylated species most abundant (data not shown). The locations of the phosphorylated residues are unknown and may differ between molecules. Figure 1C shows that this P protein competed with an IC50 of 180 nM (Kd ~90 nM). Interestingly, this is only about 4-fold greater affinity than was measured for the doubly phosphorylated C-terminal peptide D(pS)DNDL(pS)LEDF. This observation is consistent with the hypothesis that the interaction of P with N is largely, if not entirely, determined by the last several residues at the C-terminus of P.
The P C-terminus-derived peptides were tested for their ability to compete with soluble N(13-391) binding to immobilized P using SPR. The SPR results are consistent, in terms of rank order of potencies, with the results from the fluorescence anisotropy measurements, although the IC50s are higher for the SPR measurements (Figure 2 and Table 1). This is likely due to the higher ionic strength used in the SPR experiments (see Figure 1B). The SPR results demonstrate that the fluorescence anisotropy assay of the inhibition by peptides of BP5 binding to N(13-391) is a reliable surrogate for the inhibition by peptides of N(13-391) binding to P.
Inhibition of N(13-391) binding to immobilized P protein by D(pS)DNDL(pS)LEDF peptide measured by SPR. Concentrations of the peptide are indicated on the figure. The IC50 (curve shown in inset) was 13.7 ± 0.3 μM (best-fit value ± standard error of the best-fit value). The concentration of N(13-391) was 100 nM. Injection spikes have been deleted from the image for clarity.
The importance of the C-terminal Phe residue for N binding was studied by measuring the ability of the P-derived C-terminal dipeptide Asp-Phe to compete with BP5 binding to N(13-391). The IC50 was 1.45 mM (Table 1). Two-dimensional NMR experiments with 15N-labeled N(31-252) confirmed that this dipeptide binds to N(31-252), as shown by the shift in position of several crosspeaks (Figure 3A). When the longer peptide D(pS)DNDL(pS)LEDF was used, additional crosspeaks shifted position (Figure 4). This result demonstrates that the longer peptide makes more contacts with the P binding site of N(31-252) than the dipeptide. The Kds of Asp-Phe and D(pS)DNDL(pS)LEDF measured by NMR were 2.6 mM and 74 μM, respectively (Figure 3B). These Kds were higher than those measured by the fluorescence anisotropy assay because of the higher ionic strength of the buffer in which the NMR experiments were performed. As expected, this ionic strength effect was greater for the more highly charged peptide D(pS)DNDL(pS)LEDF than for Asp-Phe.
Binding of Asp-Phe (left) and D(pS)DNDL(pS)LEDF (right) to N(31-252). (A) Contour plots of backbone amide resonances of 0.15 mM 15N-labeled N(31-252) obtained from two-dimensional [15N, 1H] TROSY spectra recorded at a range of ligand concentrations. Numbered resonances correspond to the graphs in panel B. Dotted arrows denote the putative directions of shift for resonances 1 and 7. The missing resonances of 1 and 7 in the presence of 0.1 mM D(pS)DNDL(pS)LEDF indicate the binding interaction is in the intermediate exchange regime . (B) Chemical shift perturbations Δδ(N, H)obs of selected amide resonances of 15N-labeled N(31-252) as a function of ligand concentration. The symbols represent the experimental data and the lines are global best-fit theoretical curves for a simple binding isotherm. The best-fit values ± standard error of the fit of Kds were 2.6 ± 0.3 mM for Asp-Phe (left) and 74 ± 4 μM for D(pS)DNDL(pS)LEDF (right).
Backbone amide resonances of 15 N-labeled N(31-252) from two-dimensional [ 15 N, 1 H] TROSY spectra. Blue, no ligand. Red, peptide ligand. The concentration of N(31-252) was 0.15 mM. (A) 2 mM Asp-Phe. (B) 0.3 mM D(pS)DNDL(pS)LEDF.
Using the relationships ΔG = RT ln Kd and IC50 = 2 × Kd, we can obtain approximate values for ΔG of binding for the components of the C-terminal peptide of P to RNA-free N(13-391) in 10 mM Tris-HCl (pH 7.5) at 21°C. This calculation shows that Asp-Phe (DF) contributes -3.8 kcal/mole out of -8.7 kcal/mole for D(pS)DNDL(pS)LEDF. The contribution of the phenyl ring of the Phe residue can be estimated by comparing ΔG of binding for D(pS)DNDL(pS)LEDF (-8.7 kcal/mole) to that for D(pS)DNDL(pS)LEDA (-6.0 kcal/mole), hence ΔΔG = -2.7 kcal/mole, corresponding to a Kd for the phenyl ring of 10 mM. The ΔG of binding attributable to DSDNDLSLE can be estimated by subtracting the ΔG of binding of DF (-3.8 kcal/mole) from the ΔG of binding of DSDNDLSLEDF (-6.9 kcal/mole) to obtain a value of -3.1 kcal/mole for this part of the C-terminal peptide, which contains 5 acidic side chains (not counting the C-terminus) that could interact with basic residues in the P binding site of N(13-391). The ΔG of binding contributed by phosphorylation of both Ser residues can be estimated by comparing ΔG of binding for DSDNDLSLEDF (-6.9 kcal/mole) to that for D(pS)DNDL(pS)LEDF (-8.7 kcal/mole), hence ΔΔG = -1.8 kcal/mole. Interestingly, these estimates indicate that the phenyl ring of the C-terminal Phe residue contributes more free energy of binding than both phosphate moieties, that over 40% of the free energy of binding of D(pS)DNDL(pS)LEDF comes from the DF dipeptide, and that over 90% of the free energy of binding of the full-length P used in this experiment can be attributed to the D(pS)DNDL(pS)LEDF peptide. These calculations are oversimplified by not taking all factors into account, such as the fact that DF and D(pS)DNDL(pS)LEDF have free N-termini that are parts of amide bonds in the longer peptides and the full-length protein, respectively, and the unknown phosphorylation status of the C-terminal serine residues of the full-length P. Nevertheless, the calculations are helpful in demonstrating that most of the affinity of P for N(13-391) is due to the last several residues at the C-terminus of P, and that much of the affinity of these C-terminal residues is due to the binding of the last 2 residues. This observation is consistent with the idea that the N-P interaction can be considered to be a protein-protein interaction hotspot.
The relatively small energetic contribution of Ser 232 and Ser 237 phosphorylation to the affinity of D(pS)DNDL(pS)LEDF for binding to N(13-391) (-1.8 kcal/mole), combined with the observation that phosphorylation of either of the serine residues has nearly as much effect on the affinity as phosphorylation of both, suggests a possible explanation for the inconsistent results reported in the literature on the importance of Ser 232 and Ser 237 for RSV transcription and replication (see Introduction). The extent to which P C-terminal phosphorylation affects RNP assembly will depend on the local concentrations of the P and/or N proteins. If at least one of these concentrations is well in excess of the N-P binding Kd when P is unphosphorylated, then the interaction will not be significantly affected by phosphorylation. In contrast, if the concentrations of both of the proteins are lower than the Kd, then phosphorylation, by lowering the Kd, will drive the binding interaction. It may be that the N and P concentrations present in the various reported experiments differed sufficiently for the extent of P C-terminal phosphorylation to affect the efficiency of RNP assembly differently between them. For example, Mazumder and Barik  used in vitro reconstituted RNP, whereas Lu et al.  used infected cells.
In a comparison of the sequences of residues 231-241 in 163 publicly available sequences of RSV P protein in PubMed, the 3rd-to-last residue, Glu 239, is frequently conservatively replaced by Asp, and there is one instance each of Asn 234 being replaced by Asp and Ser 237 being replaced by Leu. The other residues are invariant. Similarly, the residues of N protein identified by Galloux et al.  as being involved in binding to P - Lys 46, Met 50, Ile 53, Ser 131, Arg 132, Tyr 135, Arg 150, and His 151 – are completely conserved among 126 published full-length N sequences in PubMed. This high degree of sequence conservation is consistent with the importance of P residues 231-241 and the aforementioned N residues for binding of P and N.
Three biophysical methods were used in this research: (1) a fluorescence anisotropy-based binding assay to measure competition by P-derived peptides and full-length P with binding of BP5 (D(pS)DNDL(pS)LEDF labeled on the N-terminus with BODIPY FL) to N(13-391), (2) surface plasmon resonance (SPR) to measure the competition by P-derived peptides with binding of N(13-391) to P, and (3) 2-D NMR to measure direct binding of Asp-Phe D(pS)DNDL(pS)LEDF to N(31-252). Each of these methods is suitable for screening compounds for binding to the N-P protein-protein interaction hotspot. Because of their relatively low throughput, SPR and NMR are most useful for screening small libraries of hundreds of very low molecular weight (<300 Da) fragments. The fluorescence anisotropy assay can be used to screen much larger libraries consisting of hundreds of thousands of compounds because it is a high-throughput assay. The active samples from such a screen could then be counterscreened to remove false positives due to detection artifacts using SPR and/or NMR. Thus the methods used here could be used to begin a drug discovery program aimed at identifying competitive inhibitors of the binding of RSV P protein to N that work by occupying the P binding site of N.
Oligopeptides corresponding to residues 231-241 of RSV P protein, including phosphorylation, were synthesized and purified by Biopeptide Co. Inc. (San Diego, CA). Purities of peptides were at least 95%. N- and C-termini were uncapped. Asp-Phe was from Sigma.
One μmole (1.3 mg) of peptide 1 (DSDNDLSLEDF) and 10 μmoles (5 mg) of BODIPY FL sulfosuccinimidyl ester (SSE) (Life Technologies) were dissolved in 100 μl of fresh 0.1 M NaHCO3 and reacted for 1 hour in darkness at room temperature. Peptide 5 (D(pS)DNDL(pS)LEDF), where (pS) is phosphoserine, (1.2 mg, 0.84 μmole) and 10 μmoles of BODIPY FL SSE were dissolved in 400 μl of fresh 0.2 M NaHCO3 + 50 μl of dimethyl sulfoxide and reacted for 2.75 hours at room temperature. In each case, the solution was applied to a 1 × 18-cm column of Sephadex LH-20 (Sigma) equilibrated with water. The column was eluted with water and fractions were collected. Liquid chromatography-mass spectrometry (LC-MS) was used to select fractions having the expected mass for the labeled peptide (1543 Da for BP1, 1701 Da for BP5) and little or no unlabeled peptide. For BP5, LC-MS also showed evidence of a 1682 Da species, suggesting loss of a fluorine atom, probably during the analysis. The concentration of the peptides was determined by the BODIPY FL absorbance at 504 nm using an extinction coefficient in water of 91,000 M-1cm-1.
The LC-MS analysis was performed on a Triple TOF 5600+ (AB Sciex, Redwood City, CA) equipped with a DuoSpray Ion Source and a Shimadzu LC 20-AD HPLC system (Shimadzu Scientific Instruments, Marlborough, MA). Separation was achieved on a 2.1 x 30 mm XBridge C18 column (Waters, Milford, MA) with a gradient of acetonitrile (5-90%) in 0.1% formic acid for 3 min following 1 min at 5% acetonitrile at a flow rate of 0.4 ml/min and column temperature of 30°C. LC-MS data was acquired in the TOF MS mode for m/z + from 100 to 2000. Nebulizer gas (GS1), heater gas (GS2), and curtain gas were set at 60, 70, and 30 psi, respectively. The source temperature was 600°C. Ion spray voltage was 5500 V. Declustering voltage was 100 V.
Samples of 10 μl containing peptides and proteins as indicated were prepared in buffer composed of 10 mM Tris-HCl (pH 7.5) +0.01% Brij-35 detergent (Pierce Surfact-Amps-35, Thermo Fisher Scientific, Rockford, IL) in low-volume 384-well black polystyrene assay plates (Thermo Fisher Scientific, Hudson, NH). After a 5 minute incubation, fluorescence anisotropy was measured with a Pherastar plate reader (BMG Labtech, Cary, NC) equipped with a filter module having a 485 nm excitation filter and 520 nm emission filters with polarizers. The focal height was 10.8 mm. Each measurement used 50 flashes. The parallel and perpendicular detector gains were set to 392 and 366 in order to produce an anisotropy of about 0.03 in the absence of binding.
where y is the anisotropy, y o is the anisotropy of peptide alone, [N] is the RSV-N(13-391) concentration, and y o + a is the anisotropy of fully bound peptide. The Kds were not corrected for 18% and 14% decreases in the fluorescence intensities of BP1 and BP5, respectively, upon binding.
where [C] is the competitor concentration and b is the Hill slope.
All fluorescence anisotropy measurements were performed at ambient temperature, approximately 21°C.
SPR experiments were performed on a Biacore T200 using Series S NTA sensor chips (GE Healthcare). His10-RSV P protein was immobilized on the surface using the nickel-capture amine-couple approach. The immobilization buffer consisted of 10 mM HEPES-NaOH (pH 7.5), 150 mM NaCl, 0.1 mM EDTA, 1 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and 0.005% (v/v) Tween-20. The surface was activated for His-tag capture by injecting 500 μM NiSO4 for 60 s at a flow rate of 30 μl/min. Subsequent activation for amine coupling was achieved by injecting a mixture of 200 mM 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide and 50 mM N-hydroxysuccinimide (NHS) for 7 min at flow rate 10 μl/min. His10-P protein was diluted in immobilization buffer to a final concentration of 6 μM. The protein was immobilized to ~6000 resonance units (RU) by applying short (1-4 μl) pulses of protein at 5 μl/min until the desired immobilization level was achieved. Residual NHS was blocked by a 7 min injection of 0.1 M Tris-HCl (pH 8.0) at 10 μl/min. Finally, the nickel was stripped from the surface using a 60 s injection of 350 mM EDTA at a flow rate of 30 μl/min.
Inhibition assays were carried out in 20 mM HEPES-NaOH (pH 7.5), 100 mM NaCl, 1 mM EDTA, 1 mM TCEP, 0.005% (v/v) Tween 20 and 1% (v/v) DMSO at a constant concentration of 100 nM RSV N(13-391). Peptides were mixed with N(13-391) in a 9-point concentration series of 2-fold dilutions from a top concentration of 100 μM. Three blanks were included for determining the maximum binding response. Samples were allowed to come to equilibrium for 30 min before injection at 40 μl/min for 60 s followed by a 180 s dissociation period. To monitor surface integrity, positive and negative controls consisting of 100 nM RSV N(31-252) and assay buffer, respectively, were run every 12 cycles. To correct for DMSO mismatches, three cycles of 8 solvent correction samples in the range 0-2% (v/v) DMSO were run at the beginning, middle and end of the experiment.
Data were analyzed using the Biacore T200 Evaluation Software V 2.0. Sensorgram data was reference-subtracted and solvent-corrected before the binding report point was extracted for each concentration at a time 4 s before the end of the injection. These data were used to calculate the percent inhibition according to the following equation: %Inhibition = 100*(1 – Response(conc)/Response_max), where Response_max is the average report point extracted from the 3 blank runs. Percent inhibition was plotted versus concentration and fit to a 4-parameter logistic Hill Equation (see above) for determining the peptide IC50.
NMR spectra were acquired at 298 K with a 600 MHz NMR instrument (Bruker, Billerica MA) equipped with an AVANCE III console and a triple-resonance cryogenic probe. Samples were at pH 7.5 in a 25 mM HEPES buffer containing 100 mM NaCl, 1 mM dithiothreitol, and 1 mM EDTA. The concentration of 15N labeled RSV N(31-252) was 0.15 mM. Two-dimensional [15N, 1H] transverse relaxation optimized spectroscopy (TROSY) experiments were recorded with increasing ligand concentration . Evolution times were approximately 53 ms in the 1H dimension and 29 ms in the 15N dimension. The total acquisition time was 28 min per experiment.
where Δδ(N, H)max is the maximum chemical shift difference, ET is the total concentration of N(31-352) in the solution, and LT is the total concentration of ligand.
Plasmid DNA purification, PCR product purification, and gel extraction were performed using the QIAprep Spin Mini Plasmid Purification (Qiagen Inc., Valencia, CA), QuickStepTM2 PCR Purification Kit (EdgeBio, Gaithersburg, MD), and Rapid Gel Extraction Kit (Marligen Biosciences, Ijamsville, MD), respectively. Restriction enzymes and rAPid Alkaline Phosphatase were from Roche Applied Science (Indianapolis, IN). Primers for PCR DNA were from Eurofins MWG Operon (Huntsville, AL). All PCRs were performed with High Fidelity PCR Master (Roche Applied Science) using reaction conditions specified by the manufacturer. All ligation reactions were performed using the Rapid DNA Ligation Kit (Roche Applied Science) according to the manufacturer's instructions.
A DNA sequence codon optimized for expression in E. coli, encoding N-terminal His6-tagged RSV N(13-391) [strain A2, UniProtKB/Swiss-Prot: NCAP_HRSVA, P03418] was synthesized and cloned into pET-28b (GenScript, Piscataway, NJ). The plasmid was named pJT1128.
An expression plasmid was constructed for a N-terminal His6-tagged RSV N(31-252) as follows. A tobacco etch virus (TEV) protease cleavage recognition site was placed before the His6 tag for optional removal. This was accomplished by amplifying the gene encoding residues (31-252) by PCR from the codon-optimized sequence (see above for N(13-391)) using primers 31252For (5'-GGATTCATATGGATTCCCATCGACACCCCGAACTAC-3GATTCCCATCGACACCCCGAACTAC-3') and 31253Rev (5'-GATGATGTCGACTTAGCCATACGCGTTCATGAACAGACCAGCAA-3'). The purified PCR product was digested with NdeI and SalI and ligated into a modified pET-28b plasmid to make plasmid pJT1324. The ligation mixture was transformed into E. coli DH5α chemically competent cells (Life Technologies, Grand Island, NY) and transformants were selected on Luria-Bertani (LB) agar supplemented with 30 μg/ml kanamycin. Transformants were analyzed by PCR, and plasmid DNA was isolated and sequenced for verification.
The gene sequence encoding a GST-tagged RSV P(1-241) construct was synthesized with codon optimization for expression in E. coli and cloned into pET-30a (BlueSky Bioservices, Worcester, MA). The plasmid was designated pJT1200.
The full length coding sequence (residues 1-241) for the human respiratory syncytial virus phosphoprotein P [strain A2, UniProtKB/Swiss-Prot: PHOSP_HRSVA, P03421] was synthesized in vitro by GeneArt AG (Life Technologies). The sequence was codon-optimized for Spodoptera frugiperda expression. The synthesized DNA fragment was restriction-cloned into a modified version of the pFastBac1 baculovirus expression vector (Life Technologies), downstream of an N-terminal His10 tag and a TEV protease cleavage recognition sequence.
For protein production, the plasmids pJT1128 and pJT1200 were transformed into E. coli BL21(DE3)pLysS (EMD Chemicals, Gibbstown, NJ) and plated on LB medium containing 25 μg/ml kanamycin and 100 μg/ml ampicillin at 37°C overnight. A single colony of BL21(DE3)pLysS/pJT1128/pJT1200 was inoculated into a 100-ml culture of LB containing 25 μg/ml kanamycin and 100 μg/ml ampicillin and grown overnight at 37 °C. The overnight culture was diluted to OD600 = 0.1 in 4 × 1 L of LB containing 25 μg/ml kanamycin and 100 μg/ml ampicillin and grown at 30°C with aeration to mid-logarithmic phase (OD600 = 0.6). The culture was incubated on ice for 30 minutes and transferred to 18°C. IPTG was then added to a concentration of 0.5 mM. After overnight induction at 18°C, the cells were harvested by centrifugation at 5,000 g for 15 min at 25°C. Cell pastes were stored at –20°C.
The frozen cell paste from 4 L of cell culture was suspended in 50 ml of Lysis Buffer consisting of 50 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 5% (v/v) glycerol, and 1 EDTA-free protease inhibitor cocktail tablet (Roche Molecular Biochemical, Indianapolis IN). Cells were disrupted by French Press at 18,000 psi twice at 4°C. The crude extract was centrifuged at 150,000 g for 30 min at 4°C. The supernatant was applied at a flow rate of 2.0 ml/min onto a 5-ml HiTrap Ni2+ chelating column (GE Healthcare Life Sciences, Piscataway NJ) pre-equilibrated with Buffer A consisting of 50 mM Tris-HCl (pH 8.0), 0.5 M NaCl, and 5% glycerol. The column was then washed with Buffer A, and eluted with a linear gradient from 0 to 0.5 M imidazole in Buffer A. Fractions containing RSV N(13-391) were pooled and dialyzed against 1 L of Buffer B consisting of 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1 mM dithiothreitol, and 5% glycerol. The dialyzed sample was loaded at a flow rate of 2.0 ml/min onto a 20-ml Q-Sepharose HP (HR16/10) column (GE Healthcare Life Sciences) pre-equilibrated with Buffer B. The column was washed with Buffer B and eluted with a linear gradient from 0 to 1 M NaCl in Buffer B. Fractions containing RSV N(13-391) were pooled and concentrated to 5 ml by Amicon® Ultracel-10K (Millipore, Billerica, MA). The 5 ml sample was applied at a flow rate of 1.5 ml/min to a 120-ml Superdex 200 (HR 16/60) column (GE Healthcare Life Sciences) pre-equilibrated and eluted with buffer consisting of 50 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 1 mM EDTA, 1 mM dithiothreitol, and 5% glycerol. The fractions containing RSV N(13-391) were pooled and concentrated. The protein was characterized by SDS-PAGE and LC-MS, and the concentration was determined by the Bradford method . The final yield of purified N(13-391) was 30 mg from 4 L of cell paste. The protein was stored at –80°C. GST-RSV P(1-241) was not detectably expressed in the cells and none copurified with RSV-N(13-391). The ratio of UV absorbance at 280 nm to absorbance at 260 nm (A280/A260) was 2.2. No RNA was detected in the N(13-391) protein when 2 μg of protein was run on an agarose gel and stained with ethidium bromide.
The method for overexpression of N (31-252) was the same as for coexpression of N(13-391) and P (1-241) (see above), except using only plasmid pJT1324 and omitting ampicillin. The frozen cell paste from 4 L of cell culture was suspended in 50 ml of Lysis Buffer consisting of 25 mM HEPES (pH 7.3), 0.5 M NaCl, 5% (v/v) glycerol, and 1 EDTA-free Protease inhibitor cocktail tablet. Cells were disrupted by French Press at 18,000 psi twice at 4°C. The crude extract was centrifuged at 150,000 g for 30 min at 4°C. The supernatant was applied at a flow rate of 2.0 ml/min onto a 5-ml HiTrap Ni2+ chelating column (GE Healthcare Life Sciences) pre-equilibrated with Buffer C consisting of 25 mM HEPES (pH 7.3), 0.5 M NaCl, and 5% glycerol. The column was washed with Buffer C, and eluted with a linear gradient from 0 M to 0.5 M imidazole in Buffer C. Fractions containing N (31-252) were pooled and concentrated to 5 ml. The 5-ml sample was applied at a flow rate of 1.5 ml/min to a 120-ml Superdex 200 (HR 16/60) column pre-equilibrated and eluted with buffer consisting of 25 mM HEPES (pH 7.3), 0.5 M NaCl, 1 mM EDTA, 1 mM dithiothreitol, and 5% glycerol. The fractions containing N (31-252) were pooled and concentrated. The protein was characterized by SDS-PAGE and LC-MS, and the concentration was determined by the Bradford method . The final yield of purified N (31-252) was 135 mg from 4 L of cell paste. The protein was stored at –80°C. A280/A260 was 2.6. No RNA was detected in the N(31-252) protein when 2 μg of protein was run on an agarose gel and stained with ethidium bromide.
Uniformly 15N-labeled N (31-252) was prepared by growing cells in M9 minimal medium supplemented with 1 g/L of [15N] NH4Cl and 10 g/L glucose as the sole nitrogen and carbon sources. N (31-252) was purified as described above and stored in buffer consisting of 25 mM HEPES (pH 7.3), 0.1 M NaCl, 1 mM EDTA, and 1 mM dithiothreitol. The extent of isotope labeling was >93% 15N based on comparison of the observed mass with that expected from the sequence.
Routine S. frugiperda (Sf21) cell maintenance, generation of bacmid DNA, generation of recombinant baculoviruses, expression studies in 24-deep-well blocks and roller bottles were carried out essentially as described in , except that no gentamicin antibiotic was added to insect cell cultures. SF900II medium for Sf21 cells was from Invitrogen. Fetal bovine serum (FBS) was from PAA Laboratories.
Insect cell pellets from 2 L of culture were thawed from -80°C and resuspended by homogenization in 150 ml of Lysis Buffer consisting of 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, 2 mM tris(2-carboxyethyl)phosphine (TCEP), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 3 EDTA-free protease inhibitor tablets (Roche). Cells were lysed by sonication on ice (5 × 30-s bursts) and clarified by centrifugation at 50,000 g for 1h at 4°C. The supernatant was loaded at 0.2 ml/min onto a 2-ml column of Ni-NTA Superflow resin (Roche). The column was washed with lysis buffer followed by buffer D, consisting of 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, and 2 mM TCEP, followed by additional washes at 20 mM and 40 mM imidazole in buffer D. Bound protein was eluted with 100% buffer E (buffer D with 300 mM imidazole). Fractions containing RSV P protein based on SDS-PAGE were pooled and further purified by applying at a flow rate of 0.5 ml/min to a 120-ml Superdex 75 (XK26/60) gel filtration column (GE Healthcare Life Sciences) pre-equilibrated and eluted with buffer F consisting of 50 mM Tris-HCl (pH 7.4), 200 mM NaCl, 15% glycerol, and 1 mM TCEP. The fractions with the highest purity as judged by SDS PAGE were pooled, snap-frozen in liquid nitrogen at 1.2 mg/ml, and stored at -80°C. The final yield of purified RSV P(1-241) was ~3 mg/L of cell paste. The His-tag was removed by TEV protease cleavage with a 1:200 ratio by weight of TurboTEV (Eton Bioscience, San Diego, CA) to RSV P(1-241) overnight at 4°C. The protease was removed by adsorption to immobilized Ni2+ resin.
For the BP5 competition assay, the P protein storage buffer was replaced by repeated concentration using centrifugal ultrafiltration with a Millipore Ultrafree 0.5 unit with Biomax-10 membrane (EMD Millipore, Billerica, MA) and dilution with 10 mM Tris-HCl (pH 7.5). The protein concentration was determined by the method of Bradford  using bovine serum albumin as the standard. Serial dilutions of P were prepared in 10 mM Tris-HCl (pH 7.5) +0.01% Brij-35.
ABS conceived of the study, carried out the fluorescence anisotropy measurements, and drafted the manuscript. NG expressed and purified RSV-N proteins and contributed to drafting the manuscript. NO°C performed SPR measurements and contributed to drafting the manuscript. JH performed NMR measurements and contributed to drafting the manuscript. JT prepared expression constructs and contributed to drafting the manuscript. RFG performed LC-MS measurements and contributed to drafting the manuscript. RO, IMH, and GGS prepared RSV-P expression constructs, purified the protein, and contributed to drafting the manuscript. All authors read and approved the final manuscript.
We thank Phil Ross for mass spectrometry of P protein and Stephania Livchak for removal of the His tag from P protein. This work was funded internally by AstraZeneca.


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Seven colonies on backbone only plate.
Six colonies on negative control (cells and water only).
Many colonies on LCR experimental plates and traditional ligation experimental plates.
Picked three colonies per experimental sample.
Picked three additional colonies per experimental plate to make streak colonies from. Stored at 4C.
Loaded picked colony and pipette tip into 14ml round bottom tube with 4mL LB Amp media.
Shook overnight at 37C and 244RPM.


text:
Scotiabank Profit Gets a Boost as International Recovery Gains Steam
Kevin Orland
4:51 PM IST, 30 Nov 2021 4:09 AM IST, 30 Nov 2021 4:51 PM IST, 30 Nov 2021 4:09 AM IST, 30 Nov 2021
(Bloomberg) -- The turnaround at Bank of Nova Scotia's Latin America-focused international division is picking up steam.
The unit's adjusted profit rose 74% from a year earlier to C$614 million ($480 million) in the fiscal fourth quarter, Toronto-based Scotiabank said Tuesday. Overall profit in the three months through Oct. 31 topped analysts' estimates.
Scotiabank's international division is benefiting from the global boom in commodities prices, which is helping spur growth in business loans in the Latin American markets where the unit operates. While consumer lending in the region remains sluggish, Scotiabank is posting gains in mortgages there and has kept a tight lid on the unit's costs, reducing non-interest expenses 3.1% from the previous quarter.
"International banking is showing good signs of recovery," Scott Chan, an analyst at Canaccord Genuity Group Inc., said in an interview. "They're getting a nice commodities tailwind that benefits a lot of the countries they're in."
Total loans and acceptances for the business were up 2.2% from the third quarter, the first quarter-over-quarter gain since the third quarter of fiscal 2020. The unit should get a further boost next year as interest rate increases in those markets widen lending margins, Chan said.
The bank -- which, along with Canada's other large lenders, had been temporarily prohibited by regulators from increasing its dividend or buying back shares -- raised its quarterly payout by 11% to C$1 a share and announced a plan to repurchase 24 million in shares. That works out to about 2% of the bank's outstanding stock, and at current share prices, would cost about C$1.95 billion.
Scotiabank shares fell 0.7% to C$80.88 at 9:37 a.m. in Toronto, compared with a 1% decline for the S&P/TSX Commercial Banks Index. The bank's shares have advanced 18% this year, compared with a 27% gain for the banks index.
Scotiabank is also benefiting from Canada's hot housing market, which has spurred a boom in mortgage lending. The bank's domestic division reported an average residential mortgage balance of C$255.2 billion for the fourth quarter. That's up 4.9% from the third quarter and 13% from a year earlier.
The gain in mortgage lending was driven by stronger-than-expected originations and retention, which has been a focus for the bank in recent quarters, Dan Rees, head of the Canadian banking business, said on a call discussing earnings. That growth may begin to slow next year as the housing market cools, he said.
"Should rates rise sooner in the year, as I think many of us are expecting, we expect that to soften demand," Rees said.
Also in the earnings statement:
Net income rose 35% to C$2.56 billion, or C$1.97 a share. Excluding some items, profit was C$2.10 a share. Analysts estimated C$1.91 a share, on average.
The bank took C$168 million in provisions for credit losses. Analysts estimated C$559.6 million in set-asides, on average.
Profit in Scotia's global banking and markets unit rose 9.1% from a year earlier to C$502 million.