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Library automation is not just installation of software to computerize the book purchase or data entry of books (Cataloging) or keep track of all the issue and return inside the library for some external agency for accrediation or something.
With advancement in technology like Artificial Intelligence, Virtual Reality, Augmented Reality, etc. Library will face more challenges in comming days. Library has already facing great challenges with advancment in Digital library (ebooks, ejournals, databases, etc) access from anywhere anytime with the help of remote authentication software, ofcourse.
It is a fact that 'Google' has taken away most of the searches from the library's OPAC. Today users looks for easy and faster search results, least with validity or authenticity of the information. This is where library is strong, with it's qualified professional, who can validated the information and provide the infomration as per your requirements.
How we address these challenges
We are not against the technology, after all we are technology company but we focus on humans, librarians and the users. Every library is different and as well it's users. We interact with library professionals, interact with users. Our automation is not just for the library process, we provide suits of software to automate the library. It is not necessary for library to implement all the software in one go, but we recommend step-by-step.
We make sure the library professionals and most important library committee understand the benefits of our library auotmation suits. Our Suits of Software for library is combination of software, hardware.
With us you do not get just the software or hardware for the library but gets 20+ years of experience of library both as library working professional, who understand the pain and needs of a library professional and library technologist, who can act as bridge between you and the technology geeks.
Library Management Software - Koha
Automation is not about computerization of a standard library process into the software. This kind of computerization is done way back in 80s and 90s, today with advancement of AI, VR, AR, etc. It is important that there is a "HUMAN TOUCH TO THE INFORMATION". Just because new technology will take away users from the library, can we stop it... NO. But yes we can do more than what technology can do, human touch...
Library software should provide FREEDOM to the library professionals to focus more on users than on computerization and books. Freedom not to catalogue, Freedom not do circulations, etc But freedom to interact with users, strategic planning for improving the usage of printed or digital resources, Conducting training program on different topics, like how to do research or what is the structure of a good research paper? etc. All these can happen only when your library management software is not done a computerization of the library but rather automation.
We promote Koha which supports the followings :
Cataloging standards like MARC21, FRBR, Dublin Core, etc
UNICODE, India is land of thousands of languages
SIP2/NCIP/SIP: freedom to integrate expensive RFID system from anyone
Available on any device, anytime, anywhere*
No restriction on any type of items, categories of users, policies, etc.
Software is managed by us, data inside the software is yours, cloud or onsite installation.
Reasons for supporting Koha is not just because it is open source and support international standards, etc. But because we are different with Koha, we are more than computerization...
Footfall counting as per NAAC, AICTE, UGC requirements, if any.
Automated circulation system with no hassle of missing or lost library cards, no circulation register, no proxy issue and return, etc.
Integration with third party application for automatic fine collection or payment
Integration with campus automation software for payment for vendors or transfer of library users records
Customized spine label to replace barcode, property slip, instruction slip, shelf location, etc.
More than customization, we deliver what you want with the system.
Our Automation solutions is not based on collections, users, etc., but more on type of library, library manpower, automation level, etc.
*Cloud Services
Digital Library Management Software - Dspace
Digital – the world of 0s and 1s but beyond it is the information and knowledge.
Digital library software is next to the library automation software in many libraries. Today parent organization is creating content in the form of student's presentation, project, event photographs, etc most importantly paper presentation, articles, class lectures, class notes, question papers, etc from the faculty side.
Most important challenges of digital library software is not the digital content but managing the digital content with proper technology and right & permissions, etc. Managing digital content on a digital software is not issue, who used it? why used it? what is RoI? of the in-house digital library is more important.
There is large number of manpower invested by the library staff in the digital library, true content is free or copyright of the institutes, but arranging them, cataloging them is systematic order, etc; is a time consuming job.
How we address these issues?
As Koha we support open source digital library management software like Dspace, ResourceSpace, etc.; with the same reasons as mentioned in Koha. But in digital library we have few important issues and challenge to address, for example.
We provide exhaustive statistics for the digital usage, so that library can plan or enhance the digital library services accordingly.
We help in promoting the digital library with the help customized mobile app for users to access resources; as per the digital rights policy; anywhere, anytime.
We like to customize the software as per the parent organization setup. Document management system with document life cycle process system,etc; Video Collection System with video streaming and download, etc, Image Collection System with copyright control, etc.
We can customize the complete system as Document Processing System.
Where documents life cycle can be managed like uploading process, approval process, validation process and then storing process.
Customized features for the software can be developed to work / integrate with any third party application or hardware as per client's requirements.
Ready to find out more?
Drop us a line today for a quote!


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The 2nd monitor probably was intel d865gvhz sound to identify itself to Ubuntu refresh rates, max resolutions, etc. Do the same thing again as above and you'll probably find the monitor isn't identified. That's my guess - not sure.
Intel products are not intended for use in medical, life saving, or life sustaining applications. Intel may make changes to specifications and product descriptions at any time, without notice. Page 7 Product Ecology Statements Location of Mounting Screw Holes Installing a Processor Maintenance Menu Main MenuThis download installs the integrated audio driver version for Intel® Desktop Boards with AC '97 processing with the RealTek codec intel d865gvhz sound.
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Ak chin casino maricopa az
В этом случае назначают препараты валерианы, пустырника, комбинированные растительные препараты. Систематическое выпадение геморроидальных узлов наблюдается при хронической болезни.
Обычно они сторонятся поликлиник и больниц, предпочитают уклоняться от посещений докторов и прохождения всевозможных обследований. По одной из версий, на фоне генетической предрасположенности происходит инфекционное поражение иммунной системы, приводящее к образованию комплексов из антител и вирусов, откладывающихся в тканях организма.
Example 3: Put a value in memory location vw200, and using shifting method, move this value to the output of the PLC.
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Red Deer, Alberta 7 contributions fasino helpful votes. Wouldn't Recommend. Ak Chin is a nice property.
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Review collected in partnership with Caesars Entertainment. Date of stay: December Trip type: Traveled with friends. Chin Share. Auctionone wrote a review Jul North Las Vegas, Nevada contributions 23 helpful votes. Hotel's Favorite. Loved the hotel. When checking in we had maricopa trainees plus a supervisor and they all could not do enough to make sure we were happy.
Room was extremely nice and very clean. Bed was comfortable and rain shower was good. Hotel has a great little gym and spa. We tried two restaurants and the buffet. Both restaurants were very good. Buffet seemed skimpy but we live in Las Vegas and our buffets are hard to beat.
There was a very nice chef cooking steaks and we enjoyed them over the other entrees. There is a reason it is called gambling and while I did see people winning on the slots we were not having any good fortune.
I would definitely like to stay again at the hotel as every staff person we encountered could not do enough for us and again the room was great. Date of stay: June Rooms.Shows & Entertainment- Harrah's Ak-Chin
Sleep Quality. Responded Jul 8, We are delighted to know that you loved our hotel and had a pleasant experience at our property! We are regulars at Harrah's Ak-Chin and always enjoy ourselves.
3 thoughts on "Ak chin casino maricopa az"
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Enriqueta Eshbaugh:
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Professional Community for Car Dealers, Marketing, Advertising and Sales Leaders
Today is one of those exciting days that you don't get to experience very often – the culmination of months worth of work, coming to a head. Everyone here at ShareThis has been anticipating this release for weeks, putting in extra hours in order to get ready for this moment: the launch of our newest product, the ShareThis Stream.
We're extremely proud of our work on the ShareThis Stream and think it's going to be a valuable product for our users. At its simplest, the ShareThis Stream is a great tool for cutting through the noise of the Web using the most valuable filter you have – your friends. At its deepest, the ShareThis Stream represents a wider switch that's happening right now, as sharing becomes more than just a simple utility and moves toward being a central social action for Web users.
The ShareThis Stream is a real-time view of sharing across the Web, combining the content your friends are sharing with what people are saying about it. Users sign in through Facebook Connect and Twitter, and then can instantly see what their friends have been sharing, the comments surrounding it and Tweets related to the content. You can also dig in a bit and search for content via subject or see what else people are sharing on a specific site. For a more in-depth explanation of the ShareThis Stream, please read this post from Nick Bertrand, our director of product: http://blog.sharethis.com/2010/03/02/new-sharethis-real-time-stream/
We've also built in a little something extra for our publishers, who can sign up to get the Stream on their site through our API or widgets. Also, when a user shares something from one of our publishers' sites, they'll automatically be shown a list of related shares from that site. All of these new features are designed to drive traffic and engagement on our publishers' sites.
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Views: 74
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The Pharaoh's Tomb slot is a 5-reel game with 20 payout lines, and you can place your bets across all devices. It was delivered to the market by a casino software developers of Sheriff. The slot provides vibrant graphics.
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Echinacea Leaf Marks Help
Asked July 16, 2019, 4:58 AM EDT
Hello! Just bought 2 echinacea for the first time and neither are doing well, but in different ways. One has really strange leaf marks, all brown spotted and the brown eventually turns grey and then white. Some leaf curling as well, but the blooms seem fine so far if not extremely slow to bloom. Some brown spotting on stems as well but otherwise seems healthy. My other echinacea has the brown spots on stems but none of those other symptoms - however it seems very unhealthy, the whole thing is wilting and all the leaves and blooms even the new unbloomed buds are just drooping and falling off
1 Response
Are these still in the original pots and soil? Or wee they re-potted into bigger pots after purchase? The symptoms are similar to those displayed by plants exposed to phenoxy herbicides like Weed n Feed and Crossbow, which are used to control broadleaved weeds in lawns or brush. These products tend to volatilize if used in warm weather and can move with the air to affect near by plants. Any use of herbicides recently in the vicinity of these plants? Feel free to respond to my email directly.


text: USA (WI) – Milton SDB Church A COVID19 Moment: The blessing of partnership with our SDB Conference Posted On: July 1, 2020 This article is written by Pastor Nate Crandall of the Milton SDB Church. Hope you enjoy this picture of how God is at work in and through one of our local SDB churches participating in the SDB Church Revitalization Grant program. In a moment the ... Read More Tags: Carl Greene, church revitalization, Pastor Nate Crandall Category: Church Development, SDB Exec Blog, SDB News, Uncategorized, USA (WI) - Milton SDB Church | Comment Active Life Posted On: February 12, 2020 "Fix your eyes on Jesus" is not simply a Conference week theme, it is a theme we are called to live out daily. As Conference President Kevin Butler continues to encourage us to turn our gaze upon Jesus, I believe that this powerful quote from ... Read More Tags: Carl Greene, Patti Wethington Category: Conference News, General Council, SDB Exec Blog, SDB News, Uncategorized, USA (WI) - Milton SDB Church | Comment The Connecting Church Calls Senior Pastor and Director of Youth Ministry Posted On: September 2, 2015 On August 29 following a special business meeting to discuss the recommendation of their Pastoral Search Committee, The Connecting Church (Milton, WI) voted to call Rev. Nathan Crandall as their new Senior Pastor. Pastor Nate had been serving as Interim Senior Pastor since June and ... Read More Tags: chad ochs, nate crandall, new pastor, pastoral search, the connecting church, youth ministry Category: Church News, SDB News, USA (WI) - Milton SDB Church | Comment Crandall Appointed as Interim Lead Pastor Posted On: June 4, 2015 On May 9th, at the end of each worship service, Pastor George Calhoun announced that as of May 30, 2015 he would be stepping down as Senior Pastor of the Milton SDB Church after 24+ years of service. He will be teaching at Judson University ... Read More Tags: Connecting Church, Pastor Changes Category: USA (WI) - Milton SDB Church | Comment


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George Alley was born in Charlottetown, Prince Edward Island on 22 January 1844, son of architect Thomas Charles Alley and Anne McKay. George Alley married in 1871 Mary Trew Blatch, (1843-1897). George and Mary (Blatch) Alley had the following children: Gordon Thomas Alley, MD, (1874-1925), Evelyn Trew (1873-1938), Helen Blanche (1871-1889), and George Cyril (1876-1920).
George Alley attended Central Academy, in Charlottetown. He studied law, and was admitted to the bar in 1865. In 1868, Alley joined with Louis H. Davis to form the law firm Alley and Davis. Alley was appointed judge of the Queens County Court on 18 July 1874, possibly the youngest judge ever appointed in Canada. Published reports indicate that Judge Alley had been collecting historical and archival information for many years with the intention of writing a history of the early settlement of Prince Edward Island. This was never achieved due to his untimely death. Judge Alley was a member of the Numismatic and Antiquarian Societies of Canada and was an Executive Committee member and Corresponding Secretary of the Historical Society of Prince Edward Island which was in existence from September 1881 through September 1882. Alley was involved in the militia as a Lieutenant in the PEI Lighthorse. Alley was one of the founding members of the Historical Society of Prince Edward Island whose purpose was the collection and preservation of historical documents pertaining to PEI which were in private possession. Unfortunately, after several poorly attended meetings and without government support for the endeavour, the Society folded and its collection was offered for sale to its members. Judge George Alley died 6 May 1899.
Henry Smith of Charlottetown, (1851-1941), son of William Smith (1817-1897) and Elizabeth Alley (ca. 1822-1869). Henry Smith was a carpenter, and later Clerk of the Queen's County Court. He married Eliza Lawson of Charlottetown in 1887. Henry and Eliza Smith had the following children: Bertha L. (b. 1888), Marianne Grantham (1894-1918), Henry (b. 1889), Flora (b. 1893), Helen (b. 1897), and William Raynor (died in infancy). Henry Smith was a co-worker and distant relative of Judge George Alley. Smith was co-executor of George Alley's will along with John D. Reid, Manager of the Telephone Co. Smith was a Water Commissioner for the City of Charlottetown.
Also known as the Judge George Alley, Henry Smith collection
Title based on the creators of the collection
This collection includes 1168 textual items, plus 235 images collected by Judge George Alley and Henry Smith pertaining largely to the early history of Prince Edward Island. A large proportion of the collection deals with the history of Charlottetown and the colony of Prince Edward Island during its early years of settlement. The collection is divided into 25 series, based on the arrangement that was imposed by the donor, and possibly earlier arrangement prior to donation to the Public Archives and Records Office. Arrangement was based on provenance, subject or contents or type of document. Series order has been rearranged to reflect government documents, family papers and miscellaneous. Many of the series include government records such as bills, acts, proclamations and petitions, Journals of the House of Assembly and Legislative Council, minutes of Council and claims, proposals and reports to the government. The collection includes several series of records pertaining to a number of families and individuals including: George Alley and the Alley family, Fanning family, Wright family, Bagnall family, Isaac Smith, James Bardin Palmer, and Edward Palmer. The collection includes lantern slides, photographs, textual materials and plans. The subject of the photographs is early Charlottetown, and its residents. Photographs include streetscapes, public buildings and private residences, and portraits of city officials and clergymen. The collection also includes records pertaining to the following subjects: inns, taverns, bridges, ferries, Loyalists, schools and education, the Land Question, military, agriculture, the administration of justice, postal service, land records, Charlottetown Market House, Court House, and Province House. A number of the documents in this collection are copies of original documents, many copied at the request of Judge George Alley in pursuit of his interest in writing a history of Prince Edward Island.
It is possible that some of the materials in this collections were purchased by Judge Alley from the holdings of the Historical Society of PEI. The materials collected by George Alley were passed to his son, Dr. Gordon Alley and/or Henry Smith, who possibly added to the collection. The collection was passed from Henry Smith to his daughter, Flora (Smith) Rogers, who passed it to her daughter, Marianne (Rogers) Morrow.
Correction to Series 23 (Photographs) item list, items 106 and 126, by J. Toms, 7 August 2007.
Correction to to Series 23 (Photographs) item list, item 136 (info provided by great grandson of L. C. Owen), by J. Toms, 12 July 2011.
Revision of Series 20 (History Survey) item list to identify item previous not listed (item 317a - Circular accomanying questions for information about local history of the Island, 1876), by J. Toms, 5 March 2013.
See also: Acc2541/75 and 76 transcripts of Judge Alley's 1883 lecture "P.E. Island one hundred years ago"; Acc1002 minutes and roll of members of the Historical Society of Prince Edward Island, 1881-1882; Acc2825/34 transcribed newspaper items re the Historical Society of PEI; Acc4289 The descendants of Christopher Smith of Harome, England compiled by William M. Smith, 1993; Dictionary of Canadian Biography articles for Thomas Wright, Charles Wright, George Wright, Edmund Fanning, James Bardin Palmer, and James Bagnall
Items 23, 24, 35, 40, 41 are oversize
Series includes bills and acts of the House of Assembly, minutes and rough minutes of the Council, proclamations, and a petition of the House of Assembly to the King for an allowance to PEI from the Quit Rent Fund.
See also RG2, Legislative Council fonds, and RG3 House of Assembly fonds
"ITEM LISTING AVAILABLE - ITEMS 1-47":http://www.archives.pe.ca/finding_aids/2702s1.pdf
Items 48-52, 54-57, 59-64, 66-69 are oversize
Series includes proclamations by the Lieutenant Governor on various subjects, petitions to the Lieutenant Governor and proclamations regarding embargoes on imports and exports. Series also includes some correspondence of the Lieutenant Governor regarding imports and exports of goods.
See also RG1, Lieutenant Governor fonds, Series5, Proclamations
"ITEM LISTING AVAILABLE - ITEMS 48-90":http://www.archives.pe.ca/finding_aids/2702s2.pdf
Item 922 is oversize
Series includes Journals of the House of Assembly 1780-1818, and the Journal of the Legislative Council for 1818. Also included in the series are some acts, minutes of the Legislative Council for 1815 and the 1795 Speech from the Throne.
See also RG2 Legislative Council fonds and RG3 House of Assembly fonds
"ITEM LISTING AVAILABLE- ITEMS 922-940":http://www.archives.pe.ca/finding_aids/2702s3.pdf
Items 585, 586, 589, 595, 600, 614, 615, 617, 619, 627, 629, 630, 633-635 are oversize
Series includes documents pertaining to land transactions on PEI such as land grants, petitions for land, leases, assignments, quit rent accounts and minutes of the proceedings of the proprietors and petitions of proprietors regarding quit rents.
"ITEM LISTING AVAILABLE - ITEMS 573-636":http://www.archives.pe.ca/finding_aids/2702s4.pdf
Items 695 and 738 are oversize
Series includes documents pertaining to the administration of justice in colonial Prince Edward Island. Series includes petitions for positions and claims for payment for services, petitions and reports regarding the state of the jail, correspondence, petitions and reports regarding court cases and legal opinions.
"ITEM LISTING AVAILABLE - ITEMS 685-749":http://www.archives.pe.ca/finding_aids/2702s5.pdf
Items 438-441, 446 and 462 are oversize
Series includes muster rolls of Loyalist regiments, petitions of Loyalists for land grants, biographical information for some Loyalists, lists of Loyalists, an act to empower the Lieutenant Governor to grant lands to Loyalists and a publication re the Loyalists in Prince Edward Island.
"ITEM LISTING AVAILABLE - ITEMS 425-465":http://www.archives.pe.ca/finding_aids/2702s6.pdf
Items 327 and 350 are oversize
Series includes memorials, petitions, claims for payments, tenders and estimates regarding the establishment of inns and taverns in Prince Edward Island. The series also includes similar documents pertaining to work on bridges, ferries and roads in PEI.
"ITEM LISTING AVAILABLE - ITEMS 318-350":http://www.archives.pe.ca/finding_aids/2702s7.pdf
Items 475, 476, 514, 515, 520, and 521 are oversize
Series includes act of legislature pertaining to schools and education, lists of students of various schools, certification of qualifications of teachers, claims for payment from teachers, schedules of schools, return or report of school visitor and some documents regarding the building of individual schools.
See also RG10 Department of Education fonds
"ITEM LISTING AVAILABLE - ITEMS 466-524":http://www.archives.pe.ca/finding_aids/2702s8.pdf
Items 654, 659, 661, 666, 679, and 683 are oversize
Series includes reports of the Central Agricultural Society, Judge George Alley's notes regarding military regiments in PEI, newspaper clippings re militia, a return of the Duke of Kent's Volunteer Artillery Co., committee reports and proclamations regarding the militia. Series also includes muster rolls for 3rd Company, 4th Battalion, and Shuttleworth's Independent Forresters. This series also includes a number of transcripts of documents in the French Archives pertaining to Prince Edward Island. These include census returns, and Franquet's voyage and description of Prince Edward Island in 1752. Also included in the series are documents pertaining to government administrator Philips Callbeck and the will of his wife Ann Callbeck.
"ITEM LISTING AVAILABLE - ITEMS 637-684":http://www.archives.pe.ca/finding_aids/2702s9.pdf
Series include documents pertaining to postal service in Prince Edward Island including petitions or claims for payment for the delivery of postal services, certifications of qualifications, and proposals for postal service.
"ITEM LISTING AVAILABLE - ITEMS 903-921":http://www.archives.pe.ca/finding_aids/2702s10.pdf
Item 945 is oversize
Series includes lists and descriptions of photographs and images in series 23. Series also includes various documents pertaining to the Charlottetown Market House including accounts, claims for payment for services, and proposals for building the Market House. Series also includes information regarding the Court House in Charlottetown such as reports of conditions, estimates for alterations and repairs, and correspondence re the building of a new Court House. A petition for land from Governor Fanning is also included.
"ITEM LISTING AVAILABLE - ITEMS 941-985":http://www.archives.pe.ca/finding_aids/2702s11.pdf
Items 114, 115, 127, 142, and 174 are oversize
Series includes correspondence and agreements between members of the Fanning family including former Lieutenant Governor Edmund Fanning (1739-1812) land conveyances and agreements, power of attorney documents and other documents pertaining to the Fanning vs Coles case, documents relating to the Fanning vs Guerney case, and correspondence between Jonathan Shove and J. H. Peters, agents for Fanning.
"ITEM LISTING AVAILABLE - ITEMS 112-206":http://www.archives.pe.ca/finding_aids/2702s12.pdf
Items 240, 260, 261 are oversize
Series includes documents such as correspondence, probate records, statements, affidavits and depositions regarding the settling of the Fanning land estate. Also includes records regarding the Wood-Fanning case.
"ITEM LISTING AVAILABLE - ITEMS 207-295":http://www.archives.pe.ca/finding_aids/2702s13.pdf
Items 365, 377, and 385-387 are oversize
Series includes military appointments, commissions, instructions for surveyor Charles Wright (1782-1828). Also includes papers of Thomas Wright (ca. 1740-1812), Surveyor General including correspondence with Ward Chipman (1754-1824), astronomical observations and information pertaining to his work in Isle de Sainte Croix and documents regarding the settling of his estate. Also included in the series is a warrant for payment due to George Wright (1810-1887), Surveyor General.
"ITEM LISTING AVAILABLE - ITEMS 351-424":http://www.archives.pe.ca/finding_aids/2702s14.pdf
Items 560 and 561 are oversize
Series includes records pertaining to architect Isaac Smith (1795-1871), grandfather of Henry Smith. Included in the series are contracts, agreements, specifications, and claims for payment regarding the building of the Colonial Building (Province House), the round Market House, and St. Paul's Anglican Church in Charlottetown. Also includes Isaac Smith's diary for 1858, his obituary and a photograph of Smith. Series includes research notes and photocopies relating to Isaac Smith and the Bible Societies of Nova Scotia, New Brunswick and Newfoundland.
"ITEM LISTING AVAILABLE - ITEMS 551-572, 1168":http://www.archives.pe.ca/finding_aids/2702s15.pdf
Series includes papers of James Bagnall (ca. 1783-1855) regarding his work as Government Printer, including petitions, warrants for payment for services and appointment documents. Also includes correspondence, accounts and petition of Samuel Bagnall. Series also includes a letter to Judge George Alley from J. D. Bagnall dated 1888.
"ITEM LISTING AVAILABLE - ITEMS 525-550":http://www.archives.pe.ca/finding_aids/2702s16.pdf
Item 860 is oversize
Series includes professional correspondence and legal documents pertaining to various court cases of lawyer and public office holder James Bardin Palmer (ca. 1771-1833).
See also Acc2849 Palmer family fonds
"ITEM LISTING AVAILABLE - ITEMS 833-865":http://www.archives.pe.ca/finding_aids/2702s17.pdf
Judge George Alley was one of the founding members of the Historical Society of PEI in 1881. He acted as corresponding secretary and as such corresponded with individuals supposed to have in their possession original documents pertaining to the history of PEI, or had some knowledge of particular events in PEI history. He also corresponded with historical societies in Nova Scotia, the United States and with the Archives Department in Ottawa. This series includes drafts or copies of out-going correspondence sent by Alley, as well as replies to his correspondence. Alley also engaged, through his friends and acquaintances, individuals to make copy transcripts of documents in libraries and archives outside of Prince Edward Island that pertained to Prince Edward Island history. Some documents were copied and sent to Alley, some originals were sent, some perhaps unsolicited. Alley was in the process of gathering materials with a view to writing a history of Prince Edward Island which he was unable to complete prior to his death.
"ITEM LISTING AVAILABLE - ITEMS 986-1072":http://www.archives.pe.ca/finding_aids/2702s18.pdf
Item 102 is oversize
Series includes documents pertaining to the settlement of Judge George Alley's (1844-1899) estate. Documents include land and property deeds, correspondence, bonds, and financial records. This series also includes Alley's daybook and ledger. Series includes some published materials on John Cabot and the establishment of a railroad in Prince Edward Island.
"ITEM LISTING AVAILABLE - ITEMS 91-111":http://www.archives.pe.ca/finding_aids/2702s19.pdf
Series includes 19 questionnaires sent to elderly residents of Prince Edward Island in June 1876, by D. Currie possibly on behalf of Judge George Alley, regarding the local history of Prince Edward Island. Questionnaires may have been sent as a prelude to the formation of the Historical Society of Prince Edward Island or as an exercise in gathering information for a PEI history. The questionnaires were on printed forms including 99 questions ranging from place and date of birth of the respondent to agriculture, education, local travel, crops, wildlife, game, amusements, religion, etc. The series also includes an account of the settlement of Prince County and a listing of documents from the collection of Judge Alley.
"ITEM LISTING AVAILABLE - ITEMS 296-317a":http://www.archives.pe.ca/finding_aids/2702s20.pdf
Items 1076, 1077, 1102, and 1150-1153 are oversize
Series includes some land documents, documents pertaining to 1854 assault case of McGill vs. McEachern, report of the 1847 investigation of the Immigrants' Hospital, some election materials, some petitions for payments for services provided, some correspondence between Lieutenant Governors Fanning and Patterson and Colonial Office, correspondence of Judge George Alley regarding procuring a copy of Joseph Roake journal of his 1774 journey to PEI and a copy of the journal, indenture of apprenticeship between Down and Thomas Alley, statistical information regarding PEI and its counties.
"ITEM LISTING AVAILABLE - ITEMS 1073-1163":http://www.archives.pe.ca/finding_aids/2702s21.pdf
Item 900 is oversize
Series includes documents pertaining to the Mi'kmaq and black population in Prince Edward Island. Includes report of clerk of St. Paul's Anglican Church and claims for payment for services. Series includes passenger lists for the Rambler, Humphreys, Spencer, Isle of Skye, and the Elizabeth and Amy. Also includes an unidentified diary and a copy of Samuel Holland's description of the Island of St. John in 1765 and other miscellaneous documents.
"ITEM LISTING AVAILABLE - ITEMS 866-902":http://www.archives.pe.ca/finding_aids/2702s22.pdf
Copy prints are located in photograph binders in Reading Room
Series includes 235 photographs and lantern slides of views of early Charlottetown such as streetscapes, public buildings including Province House, Fanningbank, Charlottetown City Hall, the post office, the market buildings, the court house, Central Academy, churches, and private residences including Sidmount House. The series also includes a number of portraits of prominent Charlottetown residents, public officials, clergyman, etc.
See also Series11 (items 941-943), listings and descriptions of images and 1164, 1165, 1167
"ITEM LISTING AVAILABLE":http://www.archives.pe.ca/finding_aids/2702s23.pdf
Series includes unidentified balance sheets, and day books and ledgers of unidentified general merchants.
"ITEM LISTING AVAILABLE - ITEMS 602-609":http://www.archives.pe.ca/finding_aids/2702s24.pdf
Items 768, 773, 775, 792, 797, 799, 811, 812, 821, and 824 are oversize
Series includes some correspondence and petitions regarding the Lieutenant Governor's office, petitions, warrants and claims for payments for services provided, census returns for 1810 and 1827, 1790 petition against Governor Patterson regarding his conduct, appointment and commission documents, statements regarding conditions of the hospital, grant of land for burial ground, 1816 petition regarding restoring Prince Edward Island to Nova Scotia, and copies made for Judge George Alley of correspondence between Governor Patterson and the Secretary of State for the Colonies.
"ITEM LISTING AVAILABLE - ITEMS 750-832, 1166":http://www.archives.pe.ca/finding_aids/2702s25.pdf


text:
Rappers Meek Mill and Nicki Minaj just rekindled their dirty beef and they come for each other again with several damaging allegations.
It all started when Meek liked a meme mocking Nicki's husband, Kenneth Petty, and it angered the female rapper who unleashed her tongue arsenal against Meek.
According to her: "U do IT for likes. #TwitterFingers beat women, scared of men," and continuing, she wrote: "You beat your own sister and taped it. Spit on her & taped it. Kicked me in front [of] your mother and sent her to the hospital. Sucking drake d**k made u feel tough again. Move on."
Meek quickly jumped on the fight proper, accusing Nicki of paying for the legal services of her brother, Jelani Maraj who was recently found guilty of sexually assaulting a minor, in spite that she is aware of Jelani's crime. And making several other damaging allegations against Nicki.


text:
Epacadostat Plus Pembrolizumab in Patients With Advanced Solid Tumors: Phase I Results From a Multicenter, Open-Label Phase I/II Trial (ECHO-202/KEYNOTE-037).Mitchell TC , Hamid O , Smith DC , Bauer TM , Wasser JS , Olszanski AJ , Luke JJ , Balmanoukian AS , Schmidt EV , Zhao Y , Gong X , Maleski J , Leopold L , Gajewski TF
J Clin Oncol.2018 Sep 28 ; ():JCO2018789602.PubMedで表示
スターを付ける (13view , 0users)
Tumors may evade immunosurveillance through upregulation of the indoleamine 2,3-dioxygenase 1 (IDO1) enzyme. Epacadostat is a potent and highly selective IDO1 enzyme inhibitor. The open-label phase I/II ECHO-202/KEYNOTE-037 trial evaluated epacadostat plus pembrolizumab, a programmed death protein 1 inhibitor, in patients with advanced solid tumors. Phase I results on maximum tolerated dose, safety, tolerability, preliminary antitumor activity, and pharmacokinetics are reported.
PMID: 30265610 [PubMed - as supplied by publisher]
PMID: 30265610 [PubMed - as supplied by publisher]
- Safety and clinical activity of pembrolizumab for treatment of recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-012): an open-label, multicentre, phase 1b trial.
- First-in-Human Phase 1 Study of the Oral Inhibitor of Indoleamine 2,3-dioxygenase-1 Epacadostat (INCB024360) in Patients With Advanced Solid Malignancies.
- A randomised, open-label, phase 2 study of the IDO1 inhibitor epacadostat (INCB024360) versus tamoxifen as therapy for biochemically recurrent (CA-125 relapse)-only epithelial ovarian cancer, primary peritoneal carcinoma, or fallopian tube cancer.
- Standard-dose pembrolizumab in combination with reduced-dose ipilimumab for patients with advanced melanoma (KEYNOTE-029): an open-label, phase 1b trial.
- [Review] Inhibiting IDO pathways to treat cancer: lessons from the ECHO-301 trial and beyond.


text:
H3: 08/19/11 Tarzana Joe
Fri, Aug 19, 2011 0 Comments
08191103 Hugh Hewitt: Hour 3 – After catching up on the rest of the news of the day, and discussing it with callers, guest host Guy Benson concludes the week in verse with an ode to the vacation by Tarzana Joe.
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text:
Kati by Anu Pennanen
Experimental film, 2021
Authors: Anu Pennanen, Stéphane Querrec
Produced by Palo Productions
Status: in shooting, in production
Synopsis: Part architectural documentary and part fragmented theatrical drama, it follows a fifty years old career woman struggling to establish a connection with the city she works and lives in, Espoo in Finland, and her attempt to escape into a hidden bunker out of town. Full of gaps, ellipses and episodic constructions, the fragmented narrative mirror the frustratingly abstract and normative nature of the city with an a-historical, worldless and brutal state of modernity. In this way the project articulates the vacant dreams of control with the possibility of disjunction and the uncanny.
Partners: AVEK, TAIKE, Jyväskylä Art Museum


text:
Myrtle Beach, SC
Garo Keushgerian, 42, died Monday, February 3, 2020. Born October 21, 1977 in Beirut, Lebanon, son of Anahid Kantarjian Keushgerian and the late Manouk Keushgerian.
Survivors, in addition to his mother of Myrtle Beach, include a sister, Carla Walsh; aunts and spouses, Mr. and Mrs. Mike and Sossy Megerdichian, Mr. and Mrs. Raz and Maral Banosian, and Mr. and Mrs. Robert and Salpi Westhaver; and cousins, Dr. Christine (Seepan) Parsegian, Shant (Kara) Banosian, Shiraz (Sara) Banosian, Raz Banosian, & Kyle & Derek Westhaver. He was preceded in death by his beloved grandparents, Garo and Prapion Kantarjian.
A Mass of Christian Burial will be celebrated at 11:00 AM Friday, February 7, 2020 at St. Andrew Catholic Church.
Send messages to the family using the Guestbook tab above.


text:
The lipid raft membrane has been shown to be the site of hepatitis C virus (HCV) RNA replication. The mechanism of formation of the replication complex is not clear. We show here that the formation of the HCV RNA replication complex on lipid raft (detergent-resistant membranes) requires interactions among the HCV nonstructural (NS) proteins and may be initiated by the precursor of NS4B, which has the intrinsic property of anchoring to lipid raft membrane. In hepatocyte cell lines containing an HCV RNA replicon, most of the other NS proteins, including NS5A, NS5B, and NS3, were also localized to the detergent-resistant membranes. However, when individually expressed, only NS4B was associated exclusively with lipid raft. In contrast, NS5B and NS3 were localized to detergent-sensitive membrane and cytosolic fractions, respectively. NS5A was localized to both detergent-sensitive and -resistant membrane fractions. Furthermore, we show that a cellular vesicle membrane transport protein named hVAP-33 (the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein), which binds to both NS5A and NS5B, plays a critical role in the formation of HCV replication complex. The hVAP-33 protein is partially associated with the detergent-resistant membrane fraction. The expression of dominant-negative mutants and small interfering RNA of hVAP-33 in HCV replicon cells resulted in the relocation of NS5B from detergent-resistant to detergent-sensitive membranes. Correspondingly, the amounts of both HCV RNA and proteins in the cells were reduced, indicating that hVAP-33 is critical for the formation of HCV replication complex and RNA replication. These results indicate that protein-protein interactions among the various HCV NS proteins and hVAP-33 are important for the formation of HCV replication complex.
Detergent-insoluble cholesterol- and sphingolipid-rich microdomains, known as lipid rafts, of cellular membranes play important roles in signal transduction (29), protein sorting (12), and the budding and assembly of enveloped viruses (1, 19, 21, 24). Human immunodeficiency virus type 1 (HIV-1) particles contain a high concentration of cholesterol and sphingomyelin (21, 33). Influenza virus also contains a large amount of detergent-insoluble complex and selects ordered lipid domains during its budding from the plasma membrane (24). Influenza virus hemagglutinin (HA) protein interacts with lipid raft directly via its transmembrane domain (25). Our recent studies indicate that lipid raft is also involved in the formation of hepatitis C virus (HCV) RNA replication complex (27).
HCV is the causative pathogen of non-A, non-B hepatitis. The positive-sense, single-stranded 9.6-kb RNA genome encodes a polypeptide of 3,010 to 3,030 amino acids that is processed by host and viral proteases into 10 structural and nonstructural (NS) proteins (8, 18). Most of the NS proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) of HCV were associated with the endoplasmic reticulum (ER) or other subcellular membranes when these proteins were expressed individually or as a polyprotein (10, 11, 20, 31). However, the newly synthesized HCV RNA and most of the NS proteins in the cells supporting HCV replicons were also localized to distinct speckle-like structures, which consist of detergent-resistant membrane resembling lipid raft (27). It is not clear how the NS proteins are assembled from the ER membranes to form a complex on these lipid raft membranes.
Previously, we have shown that HCV NS5B and NS5A proteins bind to two different domains of a vesicle membrane protein hVAP-33 (31), which shares characteristics with proteins of the syntaxin family (14). It is an integral membrane protein with an immunoglobulin G-like domain at the N terminus, a coiled-coil domain, and a hydrophobic transmembrane domain at the C terminus. A wide range of proteins involved in membrane fusion, including VAMP1 and VAMP2 (34, 35), interact with hVAP-33. Recent studies have shown that at least several soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins, such as syntaxin 1A, syntaxin 3, and VAMP2, are lipid raft associated (4, 12, 13). hVAP-33 also interacts with the oxysterol-binding protein (36), which binds to phosphatidylinositol and oxysterol ligands and mediates the effects of oxysterols on cholesterol homeostasis (30). Therefore, it is highly likely that hVAP-33 may play a role in the assembly of the HCV replication complex on cholesterol-rich lipid raft.
Here we analyzed the mechanism of lipid raft association of HCV NS proteins. Membrane flotation analysis demonstrated that although the individual NS proteins were localized in the different membrane or cytosolic fractions, the NS proteins in the context of the whole complement of NS proteins (NS3-NS5) or in the HCV replicon cells were localized mostly on detergent-resistant membranes. Dominant-negative mutants or small interfering RNA (siRNA) of hVAP-33 could inhibit these associations and correspondingly inhibit HCV RNA replication. These studies therefore identified a critical vesicle membrane protein in HCV replication and suggested a possible mechanism of the formation of HCV RNA replication complex.
Cell lines, constructs, and transfection.An HCV subgenomic replicon derived from plasmid 1bneo/delS (9) was kindly provided by C. Seeger of Fox Chase Cancer Center, Philadelphia, Pa. A stable cell line Huh7-replicon, in which active HCV RNA replication takes place, was selected from G418-resistant Huh7 cell colonies after electroporation with the RNA transcribed from plasmid 1bneo/delS. Cells were maintained in Dulbecco modified Eagle medium with 10% fetal bovine serum and 0.5 mg of G418/ml.
An adenovirus vector with deletions in the E1 and E3 regions was used to generate the construct expressing the HCV NS3 to NS5 proteins by using the published procedure (17). The HCV cDNA encoding NS3, NS4A, NS4B, NS5A, and NS5B proteins was cloned from a genotype 1b isolate (pCV-J4L6S [37]) by amplifying the entire NS3 to 3′-untranslated region (UTR) region by using the Expand High-Fidelity System (Roche) and cloning it into a NotI- and XbaI-digested pAdTrack-CMV shuttle vector (Q. Biogene). A control adenovirus was made from the empty pAdTrack-CMV. The resultant constructs were linearized with PmeI and cotransformed with the adenovirus backbone vector pAdeasy-1 (Q. Biogene) into Escherichia coli BJ5183 by electroporation to achieve recombination. The adenovirus recombinants were amplified in E. coli DH10B and transfected into human embryonic kidney (HEK-293) cells by using Lipofectamine (Invitrogen). Recombinant adenoviruses were harvested from cells after three cycles of freezing and thawing at day 7 posttransfection and further propagated by serial passages in HEK-293 cells.
Plasmids pcDNA3.1-NS5B and pcDNA3-NS5A were used to express HCV NS5B and NS5A proteins in Huh7 cells, as previously described (31). The PCR-generated NS4B fragment (genotype 1a, isolate H77) with or without a Flag sequence at the N terminus was cloned into BamHI site of pcDNA3.1 (Invitrogen). The PCR-generated NS3 fragment (genotype 1b) was cloned into XhoI site of a eukaryotic vector pCAGGS (22). Plasmid pcDNA3.1-Flag-hVAP-33 was used to express Flag-tagged hVAP-33 as previously described (31). The PCR-generated truncated hVAP-33 fragments were cloned into BamHI and EcoRI sites of pcDNA3.1.
Huh7 cells on six-well plates were transfected with 2 μg of DNA by using FuGENE 6 transfection reagent (Roche). The siRNA against hVAP-33, 5′-AAAGTGAAGACTACAGCACCT-3′, and the scrambled siRNA were purchased from Dharmacon Research, Inc. (Lafayette, Colo.), and transfected with Lipofectamine2000 (Invitrogen) according to the manufacturer's protocol. Huh7-replicon cells were incubated in Dulbecco modified Eagle medium with 10% fetal bovine serum at 1 day posttransfection.
Antibodies, immunoprecipitation, and immunoblotting.The primary antibodies used in the present study included a monoclonal anti-NS5B antibody as described previously (6), a monoclonal anti-NS5A antibody (Biodesign International, Saco, Maine), a monoclonal anti-NS3 antibody (clone MMM33; Novocastra Laboratories), a monoclonal anti-tubulin antibody (Sigma), a monoclonal anti-caveolin-2 antibody (New England Biolabs), a monoclonal anti-GRP78 antibody (Stressgen), and polyclonal rat sera against hVAP-33 (31). An HCV patient serum (31) was also used to detect NS5A, NS5B, and NS4B proteins. Secondary antibodies were respective peroxidase-conjugated antibodies (American Qualex). For immunoprecipitation studies, the transfected cells were harvested at 48 h posttransfection and washed with cold phosphate-buffered saline (PBS). The samples were then collected in 1 ml of radioimmunoprecipitation assay buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 12 mM deoxycholate sodium salt, 0.1% sodium dodecyl sulfate [SDS], 1% Triton X-100, 0.2 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride). After 10 passages of the cells through a 25-gauge needle, the cell lysates were centrifuged at maximum speed in a microfuge for 5 min at 4°C, and the supernatant was collected. Immunoprecipitation was performed at 4°C in TM10 buffer (50 mM Tris-HCl [pH 7.9], 100 mM KCl, 12.5 mM MgCl2, 1 mM EDTA, 10% glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride). Cell lysates (50 μl) were incubated with 3 μl of anti-Flag M2-agarose affinity gel (Sigma) in a total volume of 300 μl of reaction buffer. After incubation overnight, beads were washed four times with TM10 buffer. The precipitates were then boiled for 5 min in Laemmli sample buffer and run on a 10% polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane (Hybond-ECL plus) and analyzed by an enhanced chemiluminescence detection method (ECL Plus; Amersham).
Immunofluorescence staining.Transfected cells were fixed with 4% formaldehyde in PBS and permeabilized by 0.1% Triton X-100. The cells were then incubated with the primary antibody and TRITC (tetramethyl rhodamine isothiocyanate)-conjugated secondary antibody.
Membrane flotation assay.The membrane flotation assay was performed as previously described (28). The cells were first lysed in 0.5 ml of hypotonic buffer (10 mM Tris-HCl [pH 7.5], 10 mM KCl, 5 mM MgCl2) and passed through a 25-gauge needle 20 times. The nuclei and unbroken cells were removed by centrifugation at 1,000 × g for 5 min in a microfuge at 4°C. The cell lysates were then mixed with 3 ml of 72% sucrose in low-salt buffer (LSB; 50 mM Tris-HCl [pH 7.5], 25 mM KCl, 5 mM MgCl2) and overlaid with 4 ml of 55% sucrose in LSB, followed by 1.5 ml of 10% sucrose in LSB. To identify detergent-resistant membrane, the cell lysates were treated with 1% NP-40 on ice or 1% Triton X-100 at 37°C for 1 h before being loaded onto a sucrose gradient. The sucrose gradient was centrifuged at 38,000 rpm in a Beckman SW41Ti rotor for 14 h at 4°C. One-milliliter fractions were taken from the top of the gradient, and each was concentrated by passage through a Centricon YM-30 filter unit (Millipore, Bedford, Mass.). The sample was resuspended in SDS sample buffer and analyzed on a 4 to 15% gradient polyacrylamide gel containing SDS (Bio-Rad Laboratories, Hercules, Calif.).
Northern blot analysis.Total cellular RNA was extracted with TRIzol reagent (Gibco-BRL). Ten micrograms of total RNA was denatured by glyoxal-dimethyl sulfoxide treatment, electrophoresed through a 1% agarose gel in 10 mM sodium phosphate (pH 7.0), transferred to a nylon membrane, and immobilized by UV cross-linking. RNA was glyoxalated before being electrophoresed on the gel. An in vitro-transcribed, 32P-labeled negative-strand 3′-UTR RNA was used as a probe to detect positive-strand HCV replicon RNA. A plasmid containing the GAPDH (glyceraldehyde-3-phosphate dehydrogenase)-coding sequence was used to generate 32P-labeled negative-strand GAPDH RNA by in vitro transcription. The membrane was hybridized in Rapid-Hyb buffer (Amersham Pharmacia Biotech) for 2 h at 70°C, washed once in 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% SDS for 20 min at room temperature, twice in 1× SSC-0.1% SDS, and then twice in 0.1× SSC-0.1% SDS for 20 min at 65°C. The dried membrane was then exposed to X-ray film.
Conditional association of HCV NS proteins with detergent-resistant membrane fractions.To examine the distribution of the components of HCV replication complex, we performed membrane flotation analysis of a human hepatocyte cell line harboring an HCV RNA replicon. The cellular lysates were separated on 62-55-10% discontinuous sucrose gradients after solubilization of cells with cold 1% NP-40, a mild nonionic detergent. Under this condition, insoluble membrane proteins and lipid floated to the interface between the 55 and 10% sucrose layers, whereas cytosolic proteins and solubilized membrane proteins remained in the 62% sucrose layer. Similar to the previous report (27), a substantial proportion of NS5B and NS5A proteins was found to be associated with detergent-resistant membrane fractions (Fig. 1A and B, Huh7-replicon, fractions 1 to 5), whereas an ER marker protein, GRP78, was released to the cytosol under the same condition (28) (data not shown). The majority of NS3 protein (Fig. 1C, Huh7-replicon) was also recovered with insoluble membrane fractions (fractions 1 to 3). A similar distribution of NS proteins was observed when NS3 to NS5B proteins were expressed as polyproteins from a recombinant adenovirus (Fig. 1A to C, Huh7+Ad-NS3-5), except that the relative proportions of the various NS proteins in the detergent-resistant membranes were lower than those in the Huh7-replicon cells. In contrast, when NS proteins were expressed individually in Huh7 cells, the distributions of the individual NS proteins were significantly different. All of the NS3 was in the cytosolic fractions even without the NP-40 treatment (Fig. 1C, Huh7+NS3, fractions 6 to 9). NS5B was localized exclusively in the membrane fractions that were solubilized by the detergent (Fig. 1A, Huh7+NS5B); NS5A was present in both the detergent-resistant and the detergent-sensitive membrane fractions (Fig. 1B, Huh7+NS5A). In contrast, when a Flag-tagged NS4B was transfected alone in Huh7 cells, NS4B was completely recovered in the detergent-resistant membrane fractions (fractions 1 and 2 [Fig. 1D]). Characteristic of all of the lipid raft proteins, NS4B was almost completely solubilized by incubation with the detergent TX-100 at 37°C (Fig. 1D). These data together indicated that NS4B is the only HCV NS protein that has the intrinsic ability to associate with lipid raft. All of the other NS proteins become associated with the detergent-resistant membranes only in the presence of the complete complement of NS proteins.
NS4B formed a complex with NS5A, NS5B, and hVAP-33.Since NS4B is the only NS protein that, by itself, is localized to the detergent-resistant membrane fraction, it is likely to be the key protein that forms the core of the HCV replication complex. Thus, we investigated how NS4B interacted with the other NS proteins and cellular proteins.
We first studied the possible interaction between NS4B and NS5A. E. coli-expressed glutathione S-transferase (GST)-NS4B fusion protein was incubated with in vitro-translated, [35S]methionine-labeled NS5A and then pulled down with glutathione beads. The result showed that NS5A was pulled down together with GST-NS4B (Fig. 2A, lane 3) under the in vitro binding conditions. In contrast, NS5A did not bind GST protein under the same conditions (Fig. 2A, lane 4). As previously shown (31), NS5A interacted with GST-hVAP-33 (lane 2). We further confirmed the interaction between NS4B and NS5A by in vivo coimmunoprecipitation. NS5A was precipitated by anti-Flag antibody when it was coexpressed with Flag-tagged NS4B (Fig. 2B, lanes 1 and 2) but not in its absence (Fig. 2B, lanes 3 and 4). Furthermore, when NS5B and NS5A were cotransfected with Flag-tagged NS4B, both NS5A and NS5B were precipitated by anti-Flag antibody (lanes 5 and 6). In contrast, when NS4B and Flag-tagged NS5B were coexpressed, NS4B could not be coimmunoprecipitated by an anti-Flag antibody (Fig. 2B, lanes 7 and 8). These results combined suggest that there was no direct interaction between NS4B and NS5B; however, NS4B and NS5B could form a complex through direct or indirect NS4B-NS5A-NS5B interactions in vivo.
We next examined whether other components were necessary for the formation of the NS4B-NS5A-NS5B complex. Since NS5B binds to hVAP-33 (31), which, in turn, binds to NS5A (31) (Fig. 2A), we examined whether hVAP-33 was involved in the formation of such a complex. First, NS4B was cotransfected with Flag-hVAP-33 into Huh7 cells; the lysates were precipitated with anti-Flag antibody. The precipitates were then detected by an HCV patient's serum that recognized most of the HCV NS proteins. NS4B could not be precipitated by anti-Flag antibody, indicating that there is no direct interaction between NS4B and hVAP-33 (Fig. 2C, lanes 1 and 2). However, when Flag-tagged hVAP-33, NS5A, and NS4B were coexpressed, NS4B was coimmunoprecipitated by Flag-tagged hVAP-33 (Fig. 2C, lanes 3 and 4). The patient serum used in this immunoblot also recognized several background bands that might represent partially processed NS proteins or their degradation products (e.g., from NS5A). In contrast, when NS5B, NS4B, and Flag-hVAP-33 were coexpressed, NS4B could not be coprecipitated with Flag-hVAP-33 (lanes 5 and 6). These results combined indicate that NS5B did not interact directly with NS4B and that NS4B did not interact directly with hVAP-33. However, NS5B, NS5A, and NS4B could form a complex with hVAP-33 through a series of protein-protein interactions. This conclusion is consistent with our previous finding that hVAP-33 interacts with both NS5A and NS5B (31).
hVAP-33 is partially associated with detergent-resistant membrane fractions.The results shown above suggested that hVAP-33 may be a component of the HCV replication complex. We therefore examined whether hVAP-33 was also associated with detergent-resistant membranes. Figure 3A shows that in Huh7 cells, in the absence of detergent treatment, hVAP-33 was recovered in membrane fraction 2 only. When the cell lysates were preincubated with cold 1% NP-40 at 4°C for an hour, about half of hVAP-33 remained in membrane fractions (fractions 2 to 5), whereas the other half became soluble and retained in fractions 6 to 9. All of the hVAP-33 became soluble after treatment with Triton X-100 at 37°C. These results indicate that part of hVAP-33 is associated with lipid raft membrane. Similar distribution of hVAP-33 was seen in Huh7-replicon cells (Fig. 3B). An ER marker, GRP78, which is known not to associate with lipid raft, was completely solubilized by 1% NP-40 at 4°C. In contrast, caveolin-2, a lipid raft membrane protein (27, 29), was completely resistant to the detergent treatment at 4°C. These data suggest that hVAP-33 is present in both detergent-resistant and detergent-sensitive membranes and that its distribution is not affected by the HCV proteins. Combined with the observations that hVAP-33 binds to NS5A and NS5B, these findings strongly suggest that hVAP-33 is a component of the HCV replication complex on lipid raft membrane.
siRNA of hVAP-33 inhibited the association of HCV NS proteins with detergent-resistant membrane fractions.To establish the importance of hVAP-33 in the formation of the HCV replication complex, we first examined the effects of an siRNA against hVAP-33 in Huh7-replicon cells. The protein expression level was determined by Western blotting at 3 days posttransfection. The expression level of hVAP-33 was significantly reduced by the siRNA against hVAP-33 but not by the scrambled siRNA (Fig. 4A). As a control, the expression level of tubulin was not affected. Significantly, the level of NS5A was reduced, suggesting that HCV replication was reduced in the presence of the siRNA against hVAP-33 (Fig. 4A). To rule out the possibility that the siRNA might have induced nonspecific cytotoxic effects, we studied the growth rate of the transfected cells in the absence of G418. No differences in growth rate or percentage of cell death between the cells transfected with hVAP-33 siRNA and with scrambled siRNA were observed (data not shown), indicating that the siRNAs did not exert nonspecific cytotoxic effects. We then examined whether the distribution of NS proteins was affected by the hVAP-33-specific siRNA. As shown in Fig. 4A, the overall levels of HCV NS proteins were lower in the cells transfected with hVAP-33 siRNA. Therefore, the films for the samples that expressed hVAP-33 siRNA were exposed longer than their control samples. Figure 4B shows that most of NS5B was recovered in detergent-soluble fractions 6 to 9 in the cells expressing the hVAP-33-specific siRNA. In contrast, the distributions of NS5A and NS3 were not significantly affected (Fig. 4B). These results indicate that hVAP-33 is important for the association of NS5B with lipid raft membrane, whereas NS3 and NS5A rely on other factors for this association.
Dominant-negative mutants of hVAP-33 inhibited the association of HCV NS proteins with insoluble membrane fractions and reduced the expression level of NS5A protein and HCV RNA in Huh7-replicon cells.We further confirmed the importance of hVAP-33 in the formation of the HCV replication complex by examining the effects of the truncated mutants of hVAP-33 (as potential dominant-negative mutants) on the distributions of NS proteins. Plasmid pcDNA3.1-hVAP/1-220 encodes a truncated hVAP-33 that lacks the C-terminal 22 amino acids, which constitutes the transmembrane domain (31). Another truncated mutant hVAP-33/1-160 lacks both the coiled-coil region and the transmembrane domain. In the Huh7-replicon cells transfected with either one of the truncated hVAP-33 mutants, the relative proportions of NS5B, NS5A, and NS3 in detergent-soluble membrane fractions increased to various degrees (Fig. 5A to C). Thus, these truncated mutants served as dominant-negative mutants and inhibited the association of NS proteins with the detergent-resistant membrane, probably because they cannot integrate into vesicle membranes but retain the ability to interact with other cellular proteins associated with lipid rafts (31). The effects were particularly evident for NS5B and NS3, but less so for NS5A. Interestingly, hVAP-33/1-160, which did not bind NS5A (31), did not cause significant alteration of the association of NS5A with detergent-resistant membrane fractions, whereas it disrupted that of NS5B and NS3. Thus, the interaction between NS5A and hVAP-33 is important for the dominant-negative effects of these mutants. The overexpression of the full-length hVAP-33 did not have any effect on the distributions of NS proteins (data not shown). These results are generally consistent with the siRNA experiments (Fig. 4), except that the expression of the dominant-negative mutants of hVAP-33 had moderate effects on NS3 and NS5A, whereas the siRNA of hVAP-33 did not. Nevertheless, these data together suggested that hVAP-33 plays a role in the formation of the HCV NS protein complex.
To further confirm that the association of NS proteins with detergent-resistant membranes was important for HCV replication, we monitored the expression levels of both NS proteins and viral RNA in Huh7-replicon cells transfected with hVAP-33/1-160 or hVAP-33/1-220. The expression of the hVAP-33 truncation mutants did not affect cell growth or cell death in the absence of G418 (data not shown), which was used to maintain HCV replicon. However, when the cells transfected with these mutants were grown in the presence of 0.5 mg of G418/ml, these cells grew more slowly than the control cells without hVAP-33 transfection, suggesting a reduced production of neomycin phosphotransferase that was encoded from the HCV replicon RNA. Figure 5D shows that immunofluorescence staining of NS5A protein in the Huh7-replicon cells transfected with hVAP-33/1-160 or hVAP-33/1-220 was reduced compared to the cells transfected with hVAP-33/1-80, which expressed only the N-terminal 80 amino acids of hVAP-33 and did not interact with NS5A pr NS5B (31), or untransfected cells. The reduction of NS5A protein in these cells was probably a secondary effect of the reduced viral RNA replication, since it was not apparent until 3 to 4 days posttransfection. The hVAP-33/1-80 lacks the NS5A-interacting domain, which is localized within amino acids 81 to 160 (31); correspondingly, it did not affect the NS5A level. This result was confirmed by immunoblotting the NS5A protein (Fig. 5E). The level of tubulin served as a loading control.
To determine whether the reduced protein level correlated with the reduction of viral RNA replication, we performed Northern blot analysis of HCV RNA in these replicon cells at 3 days posttransfection. Figure 5F shows that the HCV RNA level was significantly lower in cells transfected with hVAP-33/1-160 and hVAP-33/1-220 than that in the cells transfected with the vector plasmid or hVAP/1-80. GAPDH RNA was measured as a loading control. These results confirmed that the disruption of the association of HCV NS proteins with detergent-resistant membrane correlated with the reduced viral RNA and NS protein synthesis.
In the present study we characterized the mechanism of formation of HCV replication complex on lipid raft (detergent-resistant) membrane. We showed the critical role of the interactions between the various viral NS proteins and between the NS proteins and a cellular membrane protein hVAP-33 in the formation of the HCV replication complex on detergent-resistant membranes. Lipid raft is typified by the plasma membrane caveolae, which contain caveolin-1 and caveolin-2. Caveolin-2 is a cofactor for caveola formation but is also localized on Golgi and could function in separate pathways (3). Considering that caveolae are absent in Huh7 cells (32) and that some caveolin-2 protein cofractionated with HCV NS proteins in the detergent-resistant membrane fraction (27) (Fig. 3B), these detergent-resistant membranes are most likely intracellular lipid raft, derived from the ER, Golgi, or trans-Golgi network. In addition to their association with the lipid raft, some of the NS proteins, including NS3, NS5A, and NS5B, appeared to be localized on the detergent-soluble membrane in the HCV replicon cells. Indeed, several previous reports suggested that the viral NS proteins are associated with the ER membranes (20, 23). However, these reports did not examine the detergent sensitivity of the membrane association of these proteins. Since many viral NS proteins, such as NS5A and NS3, perform multiple functions in viral replication and pathogenesis, they may be recruited to lipid raft only when participating in RNA replication. Indeed, the proportion of the NS proteins associated with detergent-resistant membrane was higher in the cells that have more active RNA replication (unpublished data). The NS proteins present in the detergent-soluble membranes (e.g., the ER) or elsewhere in the cells may be involved in other processes of viral replication, such as translation. Viral RNA molecules or the replicating RNA may also help spur the formation of the replication complex, since a higher proportion of NS proteins was associated with lipid raft in HCV replicon cells than in the cells in the absence of RNA replication (Fig. 1).
Some of the HCV NS proteins, such as NS5B and NS3, could not associate with lipid raft by themselves. Individually expressed NS3 remained in cytosolic fractions (Fig. 1C), whereas individually expressed NS5B was localized in detergent-soluble membranes. Only when they were expressed as a polyprotein did they become associated with detergent-resistant membrane, suggesting that the interactions between the HCV NS proteins are important for their lipid raft association. In the HCV replication cycle, the entire NS proteins are expressed as a polypeptide, which is then proteolytically processed by NS3-NS4A protease into individual proteins through a series of processing intermediates. The NS3-NS4A and NS5A-NS5B sites are rapidly processed, generating a more stable intermediate NS4AB-NS5A (23). Since NS4B by itself has the ability to associate with lipid raft (Fig. 1), the NS4AB-NS5A peptide is likely the peptide that initiates the formation of the replication complex on lipid raft membrane. This finding is consistent with the previous observation that NS4B by itself can induce a membrane web structure (5), which colocalized with the HCV replication complex (7). Since NS4A is a cofactor of NS3 (15, 16), NS4AB-NS5A may then recruit NS3 to lipid raft. Since NS5B is separated from NS4AB-NS5A relatively early during the polyprotein processing, the entry of NS5B into the replication complex will require additional factors. Our study shows that the cellular membrane protein hVAP-33, which interacts with both NS5A and NS5B, may perform such a role. This conclusion is supported by the finding that the relative distribution of NS5B between detergent-sensitive and detergent-resistant membranes was altered by the dominant-negative mutants and the siRNA of hVAP-33. hVAP-33 appears to play a minor role in the recruitment of NS3 and NS5A. In any case, the role of hVAP-33 in the formation of the HCV replication complex on lipid raft and in RNA replication was substantiated by the experiments with siRNA and dominant-negative mutants of hVAP-33 presented here.
The model in Fig. 6 demonstrates the presumptive steps in the formation of HCV replication complex on lipid raft. We hypothesize that soon after the HCV polypeptide is translated, NS3 and NS5B are cleaved from the polypeptide rapidly; the uncleaved NS4A-NS4B-NS5A is recruited to lipid raft by the intrinsic lipid raft-anchoring property of NS4B. NS3 then becomes associated with lipid raft by binding to its cofactor NS4A. Both NS5A and NS5B bind to hVAP-33, which is present in both the detergent-resistant and the detergent-soluble membranes, probably at the site of their synthesis on the ER. Since hVAP-33 could be localized on, and potentially shuffled between, various types of membranes in the cell, NS5B and hVAP-33 are then recruited to join NS4A-NS4B-NS5A and NS3 on the lipid raft. Since the distribution of the various NS proteins in the replicon cells was slightly different from that in the cells expressing NS3-NS5 proteins only, a replication-competent HCV RNA may also play a role in forming this complex. This diagram is consistent with the known membrane topology of the NS proteins. NS5A is a type II membrane protein, with its N terminus as a membrane anchor (2). In contrast, NS5B is a type I membrane protein (26). The C terminus of hVAP-33 binds to the N terminus of NS5A, whereas the N terminus of hVAP-33 binds to the C terminus of NS5B (unpublished). Thus, the NS5A- and NS5B-interacting domains of hVAP-33 are likely close to the membrane. NS proteins likely are enclosed by lipid raft membrane as lipid rafts coalesce. This interpretation is consistent with the finding that the NS proteins and viral RNAs in the replication complex are resistant to protease and RNase, respectively (unpublished data).
Finally, we reported here the association of hVAP-33 with both detergent-resistant and detergent-sensitive membranes. Several SNARE proteins, including the potential binding partners of hVAP-33, have been found to associate with lipid raft (4, 13, 14). The SNARE-associated lipid raft may play a role in organizing exocytotic pathways. Our finding provided an additional piece of evidence that hVAP-33 is present in specific membranes where its binding partners are localized.
We thank C. Seeger (Fox Chase Cancer Center) for providing HCV replicon1bneo/delS construct. We thank T.-Y. Hsieh for providing an NS4B construct.
This study was partially supported by NIH grants R01AI47348 and U19AI40038.
- Received 19 June 2003.
- Accepted 12 December 2003.
- Copyright © 2004 American Society for Microbiology


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The newly-appointed commissioner of the US Food and Drug Administration, (FDA) Scott Gottlieb has set out details of the agency's new digital health innovation plan, including a post-marketing approach to regulation of mobile health apps and devices.
In a blog on the FDA website, Gottlieb notes that according to one estimate, in 2016 there were 165,000 health-related apps available for Apple or Android smartphones. Forecasts predict that such apps would be downloaded 1.7 billion times by 2017.
From mobile apps and fitness trackers to clinical decision support software, digital technologies have the power to transform health care in important ways, Gottlieb says. These include:
- Empowering people to make more and better decisions every day about their own health, monitor and manage chronic health conditions, or connect with medical professionals, using consumer-directed apps and other technologies;
- Enabling better and more efficient clinical practice and decision making through decision support software and technologies, to assist in making diagnoses and developing treatment options; managing, storing, and sharing health records; and managing schedules and workflow;
- Helping to address public health crises, such as the opioid epidemic that is devastating many American communities. The FDA ran a prize competition to encourage the development of a mobile app to help connect opioid users experiencing an overdose with nearby carriers of the prescription drug naloxone for emergency treatment.
For these and other digital technologies to take hold and reach their fullest potential, the FDA needs to be forward-leaning in making sure it has implemented the right policies and regulatory tools, and communicated them clearly, to encourage safe and effective innovation. "In this rapidly changing environment, ambiguity regarding how FDA will approach a new technology can lead innovators to invest their time and resources in other ventures," Gottlieb writes.
Policies must be clear enough for developers to apply them on their own, without having to seek out, on a case-by-case basis, FDA's position on every individual technological change or iterative software development.
The 21st Century Cures Act, passed in December 2016, revised FDA's governing statute to, among other things, make clear that certain digital health technologies, such as clinical administrative support software and mobile apps that are intended only for maintaining or encouraging a healthy lifestyle, generally fall outside the scope of FDA regulation.
Such technologies tend to pose low risk to patients but can provide great value to the healthcare system.
FDA is working to implement the digital health provisions of the 21st Century Cures Act and, in the coming months, will be publishing guidance to further clarify what falls outside the scope of FDA regulation and to explain how the new statutory provisions affect pre-existing FDA policies.
The agency will provide guidance to clarify its position on products that contain multiple software functions, where some fall outside the scope of FDA regulation, but others do not.
In addition, FDA will provide new guidance on other technologies that, although not addressed in the 21st Century Cures Act, present low enough risks that FDA does not intend to subject them to certain pre-market regulatory requirements. Greater certainty regarding what types of digital health technology is subject to regulation and regarding FDA's compliance policies will not only help foster innovation, but also will help the agency to devote more resources to higher risk priorities.
In addition, FDA is planning to pilot an entirely new approach to regulating this technology, in the autumn. While the pilot programme is still being developed, the agency is considering whether and how it can create a third party certification programme under which lower risk digital health products could be marketed without FDA premarket review and higher risk products could be marketed with a streamlined FDA premarket review.
Gottlieb says certification could be used to assess, for example, whether a company consistently and reliably engages in high quality software design and testing (validation) and ongoing maintenance of its software products. Employing a unique pre-certification programme for 'software as a medical device' could reduce the time and cost of market entry for digital health technologies.
In addition, post-market collection of real-world data might be able to be used to support new and evolving product functions. For example, product developers could leverage real-world data gathered through the National Evaluation System for health Technology (NEST) to expedite market entry and subsequent expansion of indications more efficiently.
NEST will be a federated virtual system for evidence generation composed of strategic alliances among data sources including registries, electronic health records, payer claims, and other sources. The Medical Device Innovation Consortium (MDIC), is serving as an independent coordinating centre that operates NEST.
MDIC will shortly be announcing the establishment of a governing committee for the NEST coordinating centre comprised of stakeholder representatives of the ecosystem, including patients, healthcare professionals, healthcare organisations, payers, industry, and government.
FDA has been establishing strategic alliances among data sources to accelerate NEST's launch with the initial version of a fully operational system anticipated by the end of 2019.
Applying a company-based approach, rather than the traditional product-based approach, combined with leveraging real-world evidence, would create market incentives for greater investment in and growth of the digital health technology industry. Such processes could enable developers to deploy new or updated software more rapidly and would help FDA to better focus resources.


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Auction Pricedatabase
|White-Black I June 18, 2019 2PM
|was auctioned on:
|18 June 2019
BRAM BOGART (1921-2012) Blauwwit
Estimate: €1,500 - €2,000
ca. US$1,685 - US$2,247
Price realised: n. a.
BRAM BOGART (1921-2012) Blauwwit, 1964-1969. Blue and white pigment, mixed with oil. Signed and dated on the reverse 'Bram Bogart/ mei 1969'. Titled. Mounted on white painted board with annotation in pencil 'Bram Bogart/ Dec. 1964'. With inscription '44'. In the years 1964-1970 Bogart's style becomes more rigid. He returns to the geometrical forms from his earlier 'sign paintings' (cf. ), circle, square and cross, which represent for him the basic elements that can be found everywhere. Colour is the second defining element in the works from this period. Almost always two contrasting colours are brought together in one image. Bogart, who makes his own paint out of pure pigment, becomes increasingly fascinated by the power of colour itself. He chooses simple, bright colours radiating an inner strength. This interest in colour recalls Yves Klein and his quest to immaterialize art through colour. For Bogart the opposite is true: colour is matter and matter is colour. The titles are as pure and clear as the works. 'Blauwwit' (bluewhite) is nothing but a literal description of the work itself. It doesn't refer to anything else. The works are now reduced to the essential and become timeless. Roggeman writes 'Colour, form, rhythm, spiritual and material transcendence are confirmed in the art of Bram Bogart in a very personal and powerful way. But at the same time temper, intuition and spontaneity give these paintings, how basic they might seem at first glance, a rich and generous character (1). (1) W. Roggeman, Bram Bogart, Antwerpen, 1981, p. 14 ("Kleur, vorm, ritme, geestelijk en materiële transendentie (sic) worden in de kunst van Bram Bogart op een zeer eigen en krachtige wijze gehomologeerd. Maar tevens zorgen temperament, intuïtie en spontaneïteit ervoor dat deze schilderkunst, hoe primair ze op het eerste gezicht ook mag lijken, een rijke en genereuze uitstraling bezit") Dimensions: 24 x 21 cm cm (Plaat: 57 x 67 cm cm)
Informations about the auction
|Auction house:
|Veilinghuis Bernaerts
|White-Black I June 18, 2019 2PM
|Date of the auction:
|18 Jun 2019
Veilinghuis Bernaerts
Verlatstraat 16-22
2000 Antwerpen
[email protected] · +32 (0)3 2481921 · +32 (0)3 2481593
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On February 19, 1942, President Franklin D. Roosevelt signed Executive Order 9066 relocating 110,000 Japanese Americans from the West Coast into internment camps for the duration of the war. The personal rights, liberties, and freedoms of Japanese Americans were suspended by the United States Government.
After World War II in the year 1948, President Truman signed the Japanese-American Claims Act. [1] This act was a way to compensate Japanese Americans for their economic losses due to their forced evacuation. "Although some $38 million was to be paid out through provisions of the act, it would be largely ineffective even on the limited scope in which it operated." [2]
When the war ended, the American opinion of Japanese was altered. Japan was in the process of rebuilding with the help of the U.S. military. Japanese became known for their intelligence, amiable relations, and hardworking ethic. The new perspective of this country changed American minds about Japanese. In 1952, this new opinion of the Japanese resulted in first-generation Japanese Americans receiving the right to become naturalized U.S. citizens with the McCarran-Walter Act. [3]
The effort to rebuild for the Japanese Americans in America after the war was difficult because memories of imprisonment still surfaced. Many wanted justification for the harsh conditions they experienced during World War II.
"1978, the Japanese American Citizens League officially asked Congress to investigate whether the imprisonment during World War II was unjustified and wrong. A bipartisan commission conducted extensive research and, in a report titled 'Personal Justice Denied: Report of the Commission on Wartime Relocation and Internment of Civilians,' finally concluded that the imprisonment of Japanese Americans during World War II was a 'grave injustice' and resulted from 'race prejudice, war hysteria, and a failure of political leadership.'"
The Immigration and Nationality Act amendments of 1965 eliminated the national origins quota that was first established by the United States in the Immigration Act of 1924. Emanuel Celler, proposed the 1965 Act, which was strongly backed by Senator Ted Kennedy. This legislation "created the foundation of today's immigration law." [4]
"The internment of the entire Japanese-American population violated the equality principle. At the time the social movement for reparations began in the late 1970s, this principle was quite firmly entrenched in American culture, although at the time of the internment, it was not. In the America of the 1940s there was a strict racial hierarchy which was considered quite legitimate. Nevertheless, by the 1970s the organizers of the redress movement were able to make effective use of the equality principle, using it to build alliances with other groups in the U.S. dedicated to equality." [5]
Groups dedicated to the Equality Principle
"The internment also violated the principle of private property, again having more moral resonance in the 1970s than in the 1940s, when it was common to violate the property rights of nonwhite Americans. The Japanese American redress movement was also very well organized. After some internal debate and competition among various groups, the lead organization was considered the legitimate representative of the collectivity. The claimants also had access to very influential, high-level governmental insiders." [6]
"Matsunaga and Inouye were also WWII veterans. This allowed them to act as insider advocates. Inouye was also a very visible victim of violation of physical integrity, as he had lost part of his right arm in battle. The visibility of his injury became a condensation point in the struggle for reparations." [7]
The Civil Liberties Act of 1988 was an official apology made to Japanese Americans in 1988 by Congress. [8]
Financial Settlements [9]