Racing is hoping and praying the current outbreak of equine flu will not result in the abandonment of the Cheltenham Festival – as was the case in 2001.
That was the year when foot and mouth disease brought the sport to a standstill.
The cost to the industry was enormous, with the loss of Cheltenham costing the betting industry an estimated £100million alone.
With around 200,000 spectators expected at the course, the highlight of the National Hunt calendar was also worth several millions to the local economy.
The sorry saga began on February 21 when Britain's first case of foot and mouth for more than 20 years was confirmed.
Racing's fears were realised when Newcastle became the first casualty on February 26, because the course fell within an exclusion zone.
The number of confirmed cases of the disease rose steadily, resulting in racing in Britain being suspended for seven days from February 27.
Realising the severity of the situation, the majority of trainers supported the decision to suspend racing – while bookmakers called a halt to betting on races at the Cheltenham Festival.
A suspected case of foot-and-mouth at Woolstone, five miles from Cheltenham, on March 1 heightened fears for the Festival.
The racing authorities issued guidelines for re-start of racing in Britain on March 2, but Catterick and Carlisle called off meetings scheduled for the following week.
Towcester, Bangor and Wincanton abandoned their meetings on March 3.
It was after another suspected case emerged at Lambourn on March 7 that Cheltenham's hopes of racing the following week were doomed.
Plans to reschedule the meeting from April 17-19 were finally dashed on April 1 after it transpired the course was within a new exclusion zone.
There was nonetheless numb and shock when the British Horseracing Board, as it was then, announced the postponement.
Racing did get the boost it so badly needed when the three-day Grand National meeting went ahead as planned from April 5.
But it had been a long, miserable winter for thousands in and outside the industry by then.


Name of an application profile that can be used with the online resource.
Detailed text description of what the online resource is/does.
Code for function performed by the online resource.
Location (address) for on-line access using a Uniform Resource Locator address or similar addressing scheme such as http://www.statkart.no/isotc211.
Name of the online resource.
Connection protocol to be used.
Location for on-line access using a Uniform Resource Locator address or similar scheme.
Connection protocol to be used. Returns null if none.
Connection protocol to be used, or null.
Name of an application profile that can be used with the online resource. Returns null if none.
Application profile that can be used with the online resource, or null.
Name of the online resource. Returns null if none.
Name of the online resource, or null.
Detailed text description of what the online resource is/does. Returns null if none.
Text description of what the online resource is/does, or null.
Code for function performed by the online resource. Returns null if unspecified.
Function performed by the online resource, or null.


The high cost of long-term care has made planning a critically important issue for most middle class seniors and their families. In fact, most seniors will likely require some form of long-term care. Sadly, many of them are unprepared for the significant financial burdens it places on their family's hard earned savings. Financial devastation looms large for a family facing ongoing care at a rate of $8,000 - $10,000 per month.
While some seniors are able to afford private pay care, the cost of long-term care will wipe out savings of all but the wealthiest families in a matter of years. Those who have planned ahead by purchasing long-term care insurance have a degree of certainty and peace of mind, knowing that they have a lesser need to rely on other sources in the future. Unfortunately, many can't afford the high cost of long term care insurance or worse, because of age or medical condition, they cannot qualify for long term care insurance altogether. If you do have long-term care insurance, you should be aware of what your policy covers. Does your policy cover home care. Does the home care attendant need to come froma certified agency? How many Activities of Daily Living do I need assitance with before my policy begins to pay for my care? Does my policy have a cost of living increase associated with it? Is my policy a NJ Partnership Program approved policy. For many, a long term care insurance policy can ofset some of the costs of care, but may not be enough. Consequently, Medicaid may still be necessary.
While Medicaid eligibility with respect to long-term care was not difficult in the past, there has been a steady drift towards more complex and restrictive rules, the latest being the Deficit Reduction Act of 2005 which went into effect on February 8, 2006. These changes have resulted in complex eligibility requirements for those in need of Medicaid benefits. It's no longer as easy as reviewing one's bank statements. There are a myriad of regulations involving look-back periods, income caps, transfer penalties and waiting periods to plan around.
Our law firm has the experience and the expertise to help you navigate through the maze of long term care. Contact us today to start the process of understanding the issues surrounding Medicaid eligibility and to implement the planning and application process.


WASHINGTON — A composed and contrite Mark Zuckerberg held up under hours of grilling by more than 40 senators Tuesday — but his performance did little to mask Facebook's growing political problems in Washington.
"You don't think you have a monopoly?" Senator Lindsey Graham (R-S.C.) asked incredulously. Zuckerberg responded, "It certainly doesn't feel that way to me," eliciting a few laughs in the packed Senate chamber.
Senator Richard Blumenthal (D-Conn.), a former federal prosecutor, challenged Zuckerberg on how he can maintain that Facebook hasn't violated a 2011 privacy settlement it reached with the Federal Trade Commission. Senator Ted Cruz (R-Texas) pressed him on why Facebook, which conservatives have accused of political bias, should continue to receive federal protection from liability for content its users post.
And Senator John Kennedy (R-La.) made it clear he was not at all mollified by Zuckerberg's repeated apologies.
Antitrust action or tougher, European-style data laws are probably still not an immediate danger for Facebook in a federal government dominated by anti-regulation Republicans — one bill Zuckerberg endorsed, S. 1989 (115), which would require more disclosure for online political ads, is still expected to face tough sledding in Congress.
The critique was bipartisan. "We've heard you apologize numerous times and promise to change, but here we are again," said Senator Maggie Hassan (D-N.H.).
The senators seemed to struggle with the nuances of the data-driven internet in ways that took the heat off Zuckerberg. In one exchange, Senator Orrin Hatch (R-Utah) asked Zuckerberg how the company manages to make money.
"We sell ads, senator," Zuckerberg said.
But one expert in the art of congressional hearings said Zuckerberg might not be so lucky when he appears Wednesday for round two on Capitol Hill, this time in front of the House energy and commerce committee.
Li Zhou, John Hendel and Steven Overly contributed reporting.


The KÜHL Vala™ Skirt features Tufflex™ fabric, a durable woven stretch softshell with a soft brushed back for comfort. This incredible versatile fabric is lightweight, quick drying, wrinkle resistant and has a UPF 40 sun protection.
We've added mechanical stretch to our Tufflex™ for a four way stretch (mechanically on the loom and materially with Spandex fibers) for freedom of movement.
Two deep front pockets and on the back notice the embroidery detail on the two snap back pockets. There is also one large, low profile pocket at the right thigh. We even added one small stash pocket under the waistband. With a UPF 40 giving you care free sun protection. Easy wash and wear wrinkle resistant Tufflex™ fabric make the Vala™ Skirt great for travel.


text:
Tue January 21, 2020
Knick Moore Let's Get Critical
When much of your time is spent in theaters picking apart individual performances, wardrobe malfunctions, and minute scenic infractions, it's easy to forget that you're watching something that's meant to be entertaining. That in mind, this week I found myself in a preview performance of Theatre Baton Rouge's production of A Gentleman's Guide to Love and Murder experiencing something I hadn't felt in quite some time: delight.
The show has definite Gilbert and Sullivan vibes, and while it would be easy to criticize it as a pastiche, the concept is freshened with modern humor and nods to the indelicacies of its Edwardian setting.
We are led on our journey of romance and death by Lord Montague "Monty" D'Ysquith Navarro, Ninth Earl of Highhurst, who has clawed his way to his rightful title and lands by offing the eight heirs ahead of him. Played by Jonathan Thomas, Monty is on stage for the entirety of the show, and Thomas somehow finds the strength to keep up a vocal and comic balancing act until the final curtain.
This is all the more impressive considering he spends much of his time sparring with TBR veteran Albert Nolan, who plays all nine members of the D'Ysquith family in turn, giving each of them unique voices and personalities. His death scenes are humorous and numerous, and his musical numbers weave some of the best jokes into the show.
As if murdering nearly a dozen people wasn't enough, Monty also juggles not one but two romantic interests. The first is Sibella Hallward, played by Marion Bienvenu, who keeps him on his toes both amorously and vocally, and manages to make a money-grubbing adulteress somehow sympathetic.
Horror and comedy come from the same source: surprise. There were moments where the cast — both individually and as a whole — surprised with a smile, a snort, and even a string of giggles after misdirecting me before a sideswipe.
The show is full of surprises, but one of the most delightful is Brandy Cramer, making her debut on the TBR stage. The cast is a smorgasbord of intimidating talent, and she holds her own in remarkable fashion. One of the biggest grins I've experienced in quite some time came during "I've Decided to Marry You," when Thomas finds himself trapped between Bienvenu and Cramer. The trio juggle lyrics and music so precisely, any hesitation would send the piece crashing to the floor.
All of this demands much of the chorus, and they are up to the task, surviving a constantly changing set by each carrying multiple roles, accents, and wardrobe changes.
Taken together, the flawless execution of this technically demanding show makes it one you shouldn't miss. A Gentleman's Guide to Love and Murder opens Friday, November 1, and runs through the November 17. Murder has never been funnier.
Previous Dorque, Episode 214: Batmania
Awkward Company
Physical inability to experience pleasure is the only conceivable reason to explain The Advocate's George Morris' latest book report of a review for TBR's production of Stephen Sondheim's "Company."
GUEST COLUMN: college is awesomeeee! – Kayleigh Hebert, LSU Freshmannnn https://t.co/ewWpw8yPFB https://t.co/ieMEbRoxV222 hours ago
PETA Seeks to Save Endangered Crab Lice With "Push for Bush" Campaign https://t.co/0BWof40bCE https://t.co/RdcN8l7QBW2020/01/19

redpajama_set_name: RedPajamaCommonCrawl


text:
Constructibility in Quantum Mechanics
BenDaniel, David Jacob (2011) Constructibility in Quantum Mechanics. In: UNSPECIFIED.
Constructibility_BenDanielrev_(4)_-_2_Proceedings.pdf - Presentation
Available under License Creative Commons Attribution No Derivatives.
D.J. BENDANIEL
Cornell University (djb16@Cornell.edu)
Ithaca NY, 14853, USA
We pursue an approach in which space-time proves to be relational and its differential properties fulfill the strict requirements of Einstein-Weyl causality. Space-time emerges here from a set theoretical foundation for a constructible mathematics. In this theory the Schrödinger equation can be obtained by adjoining a physical postulate of action symmetry in generalized wave phenomena. This result now allows quantum mechanics to be considered conceptually cumulative with prior physics.
A set theoretical foundation is proposed in which the axioms are the theory of Zermelo-Frankel (ZF) but without the power set axiom, with the axiom schema of subsets removed from the axioms of regularity and replacement and with an axiom of countable constructibility added. Four arithmetic axioms are also adjoined; these formulae are contained in ZF but must be added here as axioms. All sets of finite natural numbers in this theory are finite and hence definable. The real numbers are countable. We first show that this constructible theory gives polynomial functions of a real variable, which are of bounded variation and locally homeomorphic. Eigenfunctions governing physical fields can then be effectively obtained. By using an integral of the Lagrange density of a field over a compactified space, we produce a nonlinear sigma model. Finally, the Schrödinger equation follows directly from the postulate and a sui generis proof in this theory of the discreteness of the space-like and time-like terms of the model.
Conference or Workshop Item (UNSPECIFIED)
BenDaniel, David Jacob djb16@cornell.edu
Foundations of Physics, Nonlinear Sigma Model, Quantum Fields
General Issues > Laws of Nature
David Jacob BenDaniel
Constructibility in Quantum Mechanics. (deposited 24 Jun 2010)


text:
Khaddafy Meets with Lockerbie Family Members in NYC
By Caitlin Millat
Published Sep 26, 2009 at 6:59 AM | Updated at 7:15 AM EDT on Sep 26, 2009
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Libyan leader Moammar Khaddafy continued his whirlwind New York City trip with a visit to the family of a man killed in the Lockerbie bombings, an attack widely attributed to the dictator Khaddafy's home country.
Khaddafy met with the sister of a man who died in the 1988 bombing of Pam Am flight 103, which crashed over Lockerbie, Scotland in an international collision of world powers that caused the U.S. and Libya to split ties until 2006.
"He generally said he was very sorry for the loss, but we didn't go into any details about the bombing," attorney Lisa Gibson said of the meeting, which took place at the Libyan Mission to the United Nations Wednesday. Gibson's brother was stationed in the Army in Berlin and was coming home for Christmas when he and 269 others were killed in the bombing.
"He was very friendly and cordial to us," said Gibson, who gave Khaddafy a pen and a card and told the dictator she had been praying for him. "Honestly, I think he was touched by us being there."
G-20 in Photos: Obama Talks, Protesters Walk
Gibson, a Libyan activist who has been three times to the country and has raised money for Libyan children with AIDS, said their meeting was arranged through a Libyan ambassador. She also said another person who had lost a family member -- their father -- in the Lockerbie bombing was present at the meeting.
Khaddafy was in town for the U.N. summit, camping out in Westchester when he was refused a room by New York City's hotels.
Convicted Lockerbie bomber Abdel Baset al-Megrahi was released last month after al-Megrahi was diagnosed with fatal prostate cancer.
The Obama Presidency in Photos


At NexGen Pharma we believe that quality is a holistic commitment and a fundamental principle in every step of the production process. We believe quality is not only founded in the end product and resulting efficacy. But that quality originates from the facility and raw materials used and extends beyond purchasing a product to post customer care service and the establishment of trust.
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Extending from our foundation built on quality, we operate with a leading team of pharmacists, compounding and dispensing technicians and customer care specialists. Our team has used their 80 years plus cumulative experience in the industry to refine a series of processes and procedures that have become our protocol to yield the highest quality medications. All our products are subject to our purpose developed quality management system to ensure they are reliably accurate, pure and free from microbial contamination.
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Regulated storage of raw materials.
All raw ingredients are subject to a quality assurance check. The ingredients are checked, weighed and stored separately in their own-labeled container. The quality and accuracy of the ingredients is checked each time.


text:
Q: Excel and Word behaving difrerently in the same code I have a problem - instances of Excel and Word behave differently in the same procedure. Have a look at the code. The idea there is to have a procedure that handles resaving files in excel and word in various format combinations.
The problem is that I notice that word and excel behave differently - the appWord and appExcel have different type names. At some point appWord is changed from Application to Object, which then makes it impossible to close it. I don't understand the differences in the behaviour, since the code applied to them is identical.
Option Explicit
Dim fso
Dim appWord
Dim appExcel
Set fso = CreateObject("Scripting.FileSystemObject")

startWord
ResaveFiles appWord.Documents, "docx", 12, 0
appWord.quit

startExcel
ResaveFiles appExcel.Workbooks, "xlsx", 51, 56
appExcel.quit

MsgBox "All done."

Sub ResaveFiles(appType, srcExtName, srcExtNum, tmpExtNum)
Dim objFile
Dim objOpenFile
Dim strDirectory
    For Each objFile in fso.GetFolder(".").Files
        If lcase(fso.GetExtensionName(objFile)) = srcExtName Then
                If typeName(appType) = "Documents" Then StartWord
                If typeName(appType) = "Workbooks" Then StartExcel
            Set objOpenFile = appType.Open(objFile.path)
            strDirectory = fso.BuildPath(objOpenFile.path, fso.GetBaseName(objOpenFile.name) & "._temp")
            objOpenFile.SaveAs strDirectory, tmpExtNum
            objOpenFile.Close
            msgBox typename(appType) & objFile
            msgBox typename(appWord) 'First typename test
            msgBox Typename(appExcel)
                If typeName(appType) = "Documents" Then appWord.Quit
                If typeName(appType) = "Workbooks" Then appExcel.Quit
            set objOpenFile = appType.Open(strDirectory)
            objOpenFile.SaveAs objFile.path, srcExtNum
            objOpenFile.Close
            fso.DeleteFile(strDirectory)
            msgBox typename(appWord) 'Second typename test
    msgBox Typename(appExcel)
        End If
    Next
    End Sub

'Start Word
 Sub StartWord
            Set appWord = CreateObject("Word.Application")
                appWord.visible = false
                appWord.DisplayAlerts = false
    End Sub

'Start Excel
Sub StartExcel
            Set appExcel = CreateObject("Excel.Application")
                appExcel.visible = false
                appExcel.DisplayAlerts = false
End Sub

I have tested it in the following way (with two typename tests) - when there are word files available, first appWord is Application and appExcel is empty, then it changes to Object and appExcel stays Empty (in this case we get an error when the subprocedure ends at AppWord.Quit). When there are no word files, and the script is processing Excels, first appWord is Object and appExcel is Application, then appWord is still Object and appExcel is still Application - in this case there are no errors when the subprocedure ends, on the appExcel.Quit.

A: Maybe i'm wrong, just my opinion:
If typeName(appType) = "Documents" Then appWord.Quit
If typeName(appType) = "Workbooks" Then appExcel.Quit

set objOpenFile = appType.Open(strDirectory)

appType is a reference to what appWord.Documents or appExcel.Workbooks are referencing before entering your ResaveFiles Sub, where you instantiate a new copy of 'Excel.Application' or 'Word.Application', and in each of the cases, you instruct the application TO QUIT. The question is not why in the case of word you got an error. From my point of view YOU SHOULD got an error. The question is why, if instructed to quit, excel keeps open and maintaining references to handle your code.
EDIT - And not tried. Just adapted from OP code. Adapt as needed
Option Explicit

    ResaveFiles "Word.Application", "docx", 12, 0
    ResaveFiles "Excel.Application", "xlsx", 51, 56

MsgBox "All done."

Sub ResaveFiles(progID, srcExtName, srcExtNum, tmpExtNum )
Dim app, doc
Dim fso, objFile, objOpenFile, strDirectory

    Set fso = CreateObject("Scripting.FileSystemObject")
    For Each objFile in fso.GetFolder( "." ).Files
        If LCase(fso.GetExtensionName( objFile.Name )) = srcExtName Then

            ' Get references
            Set app = GetNewAppInstance( progID )
            Set doc = GetDocumentHandler( app )

            ' Save temp
            Set objOpenFile = doc.Open( objFile.Path )
            strDirectory = fso.BuildPath( objOpenFile.path, fso.GetBaseName(objOpenFile.name) & "._temp" )
            objOpenFile.SaveAs strDirectory, tmpExtNum
            objOpenFile.Close

            ' Release objects
            Set objOpenFile = nothing
            Set doc = nothing
            app.Quit
            Set app = nothing

            ' Get references again
            Set app = GetNewAppInstance( progID )
            Set doc = GetDocumentHandler( app )

            ' Resave file
            Set objOpenFile = doc.Open( strDirectory )
            objOpenFile.SaveAs objFile.path, srcExtNum
            objOpenFile.Close

            ' Release objects
            Set objOpenFile = nothing
            Set doc = nothing
            app.Quit
            Set app = nothing

            ' Clean
            fso.DeleteFile(strDirectory)

        End If
    Next

End Sub

Function GetNewAppInstance( ByVal progID )
    Set GetNewAppInstance = CreateObject( progID )
    With GetNewAppInstance
        .Visible = False
        .DisplayAlerts = False
    End With
End Function

Function GetDocumentHandler( app )
    Dim name
    name = app.Name
    If InStr(name,"Excel") > 0 Then
        Set GetDocumentHandler = app.Workbooks
    ElseIf InStr(name,"Word") > 0 Then
        Set GetDocumentHandler = app.Documents
    Else
        Set GetDocumentHandler = app
    End If
End Function

redpajama_set_name: RedPajamaStackExchange


text:
Protein/Protein Interactions
A Targeted Spatial-Temporal Proteomics Approach Implicates Multiple Cellular Trafficking Pathways in Human Cytomegalovirus Virion Maturation
Nathaniel J. Moorman, Ronit Sharon-Friling, Thomas Shenk and Ileana M. Cristea
Molecular & Cellular Proteomics May 1, 2010, First published on December 20, 2009, 9 (5) 851-860; https://doi.org/10.1074/mcp.M900485-MCP200
Nathaniel J. Moorman
Ronit Sharon-Friling
Thomas Shenk
Ileana M. Cristea
For correspondence: icristea@princeton.edu
The assembly of infectious virus particles is a complex event. For human cytomegalovirus (HCMV) this process requires the coordinated expression and localization of at least 60 viral proteins that comprise the infectious virion. To gain insight into the mechanisms controlling this process, we identified protein binding partners for two viral proteins, pUL99 (also termed pp28) and pUL32 (pp150), which are essential for HCMV virion assembly. We utilized HCMV strains expressing pUL99 or pUL32 carboxyl-terminal green fluorescent protein fusion proteins from their native location in the HCMV genome. Based on the presence of ubiquitin in the pUL99 immunoisolation, we discovered that this viral protein colocalizes with components of the cellular endosomal sorting complex required for transport (ESCRT) pathway during the initial stages of virion assembly. We identified the nucleocapsid and a large number of tegument proteins as pUL32 binding partners, suggesting that events controlling trafficking of this viral protein in the cytoplasm regulate nucleocapsid/tegument maturation. The finding that pUL32, but not pUL99, associates with clathrin led to the discovery that the two viral proteins traffic via distinct pathways during the early stages of virion assembly. Additional investigation revealed that the majority of the major viral glycoprotein gB initially resides in a third compartment. Analysis of the trafficking of these three viral proteins throughout a time course of virion assembly allowed us to visualize their merger into a single large cytoplasmic structure during the late stages of viral assembly. We propose a model of HCMV virion maturation in which multiple components of the virion traffic independently of one another before merging.
Viruses are obligate intracellular parasites that rely on the host cell for many of the processes essential for their replication. Viruses subvert cellular pathways detrimental to virus growth and hijack complex cellular machinery necessary for efficient viral replication. This complex relationship with the host cell is born of necessity; viruses contain relatively little genetic material, which frequently encodes for a limited number of proteins. Understanding the molecular events at the interface of the virus-host relationship is crucial for the identification of targets for future therapeutics.
One of the most complex processes in the viral life cycle is the assembly of infectious viral particles. Multiple events must be coordinated in time and space for the correct incorporation of the viral genetic material into an infectious unit, or virion. For example, the virions of herpesviruses are composed of three essential elements: (i) the viral DNA and its surrounding protein shell, which together form the nucleocapsid, (ii) a layer of proteins surrounding the nucleocapsid termed the tegument, and (iii) a lipid bilayer, or envelope, surrounding the tegumented nucleocapsid in which viral glycoproteins are embedded (1–3). Although the formation of the nucleocapsid occurs in the nucleus, the herpesvirus tegument is acquired as the nucleocapsid moves into the cytoplasm through an undefined mechanism (2, 3). Once in the cytoplasm, the tegumented nucleocapsid acquires its envelope, again through a poorly defined event.
Although it has been known for some time that multiple HCMV1 proteins important for viral assembly localize to a juxtanuclear site termed the assembly zone, only recently have cellular markers for the HCMV assembly zone been described (4–7). This work has led to the view that HCMV virion maturation occurs by progressing through the endoplasmic reticulum to the Golgi apparatus (4, 5) and then via the ESCRT pathway to its site of release into the extracellular space (8). This work represents a major step forward in our understanding of the spatial requirements for virion maturation. However, the molecular events underlying this process remain poorly defined.
The assembly of HCMV particles is a process that has limited homology to cellular events. Therefore, it comes as no surprise that HCMV encodes proteins that are essential for the orchestration of virion assembly. One such protein, pUL99 (also termed pp28), is encoded by the UL99 ORF and resides in the cytoplasm of infected cells (9). pUL99 is required for the envelopment of capsids to generate virions (10, 11). In its absence, tegumented capsids accumulate in the cytoplasm of infected cells (11). This function may require its interaction with the cellular chaperone BiP as pUL99 binds to BiP during infection and disruption of BiP function was shown to inhibit HCMV assembly (12). Despite its critical role in envelopment, pUL99 is dispensable for cell-to-cell spread of HCMV, a process that does not require enveloped nucleocapsids (13).
Another HCMV protein integral to the assembly process is pUL32 (also termed pp150), encoded by the UL32 ORF. Like pUL99, this protein is required for the production of HCMV virions (14–17). pUL32 is capable of directly binding to the nucleocapsid, and this event occurs shortly after nucleocapsid assembly (18). Despite its localization to both the nucleus and the cytoplasm during infection (19, 20), pUL32 acts primarily in the cytoplasm where it may affect the localization and stability of the nucleocapsid (16, 21). In the absence of this viral protein, nucleocapsids are found primarily in the nucleus with few viral particles in the cytoplasm (21).
To generate additional insights into the molecular events controlling HCMV virion assembly, we used a targeted genetic-proteomic approach to identify protein binding partners for pUL99 and pUL32 in the context of viral infection. This work led to the identification of multiple distinct steps in the virion maturation process. The two viral proteins traffic through two separate compartments at the early stages of viral assembly. Although the tegument and nucleocapsid traffic with pUL32 in a clathrin-associated, brefeldin-insensitive vesicle, pUL99 localizes to a distinct brefeldin-sensitive structure that colocalizes with markers of the ESCRT trafficking pathway. The viral envelope glycoprotein B (gB), which is encoded by the UL55 ORF, did not substantially colocalize with pUL99 or pUL32 at early stages of the assembly process. However, we demonstrate that as infection progresses in time these three distinct compartments merge into a single vesicle, which may represent the final site of virion assembly.
EXPERIMENTAL PROCEDURES
Cells, Viruses, and Reagents
Primary human foreskin fibroblasts were used between passages 5 and 15 for all infections. Human foreskin fibroblasts were cultured in medium (Dulbecco's modified Eagle's medium) containing 10% newborn calf serum. The laboratory-adapted wild-type strain AD169, BADwt (23), was used to infect fibroblasts in 150-cm dishes except as indicated. For large scale immunoisolations, cells were infected for 1 h in 5 ml of Dulbecco's modified Eagle's medium + 10% newborn calf serum/dish. After 1 h, the inoculum was removed and replaced with fresh medium.
Recombinant Virus Construction
We used linear recombination in Escherichia coli (14, 22) to generate the BADinUL99GFP and BADinUL32GFP viruses in the BADwt (23) background. Briefly, PCR was used to generate a targeting cassette for recombination. The template for PCR contained a GFP tag followed by a kanamycin cassette flanked by Frt sites. The underlined sequences target the PCR product for homologous recombination into the HCMV genome. The following primers were used: UL99GFP(F), 5′-TCGTGTACACGGCCGGGGAGGGCGACGTGGTACAGATGGTGGTCGTGGTCGCCGGAAGAAGATGGAAAAAG-3′; UL99GFP(R), 5′-ACTACAAAAAAAAAAGCTGAACATGGTCATCTAGCAGCAAAGTTCTCCTTCGTCGTGGAATGCCTTCG-3′; UL32GFP(F), 5′-GCCGTGCAGAACATCCTCCAAAAGATCGAGAAGATTAAGAACACGGAGGAAGCCGGAAGAAGATGGAAAAAG-3′; and UL32GFP(R), 5′-TCACTATCCGATGGTTTCATTAAAAAGTACGTCTGCGTGTGTGTTTCTTAACGTCGTGGAATGCCTTCG-3′. The PCR product was transformed into recombination-competent DY380 E. coli, and chloramphenicol (CamR)- and kanamycin (KanR)-resistant bacteria were selected. Chloramphenicol resistance is provided by the BAC backbone. BAC DNA was purified from CamR, KanR colonies and checked for gross rearrangements by restriction digest. Recombinant BAC DNA was transformed into DH10B to alleviate concerns regarding spurious recombination in DY380. The kanamycin resistance cassette was removed by induction of flp recombinase with arabinose for 1 h at 37 °C. Bacteria were screened for loss of KanR and retention of CamR.
Immunoaffinity Purification of Virus-Host Protein Complexes
Fibroblasts were infected with BADinUL99GFP or BADinUL32GFP at a multiplicity of 3 pfu/cell. Approximately 1 g of infected cells was used per isolation (approximately 30 150-mm dishes). After 72 h, the cells were harvested and frozen in liquid nitrogen as small pellets as described previously (22). Cryogenic lysis was performed using a Retch MM301 Mixer Mill (Retch, Newtown, PA) as described (24, 25), and the resulting frozen cell powders were suspended in an optimized lysis buffer (20 mm K-HEPES, pH 7.4, 0.1 m potassium acetate, 2 mm MgCl2, 0.1% (v/v) Tween 20, 1 μm ZnCl2, 1 μm CaCl2, 1% Triton, 250 mm NaCl, 1:100 (v/v) protease inhibitor mixture (Sigma)) to yield the cell lysate. Anti-GFP rabbit polyclonal antibodies were prepared, affinity-purified, and conjugated as 5 μg of antibodies/mg of M-270 epoxy magnetic beads (Invitrogen Dynal) as described previously (24). 7 mg of conjugated beads were utilized for each immunoaffinity purification. Affinity purifications were performed by 1-h incubation of the cell lysate with magnetic beads at 4 °C, and the isolated complexes were eluted with 700 μl of a freshly made 0.5 n NH4OH, 0.5 mm EDTA solution, frozen in liquid nitrogen, and dried using vacuum centrifugation (SpeedVac Plus SC110A with gel pump GP110, Thermo Scientific) as described (24). The dried sample pellets were suspended in 30 μl of SDS-PAGE buffer with reducing agent (Invitrogen), incubated at 70 °C for 10 min, alkylated with 100 mm iodoacetamide (30 min at room temperature in dark), resolved by one-dimensional gel electrophoresis on a 4–12% NuPAGE Novex Bis-Tris gel (Invitrogen), and stained with SimplyBlueTM SafeStain (Invitrogen).
Mass Spectrometry Analyses of Co-isolated Proteins
To identify the virus and host proteins isolated with pUL99 and pUL32, the entire gel lanes from each isolation were cut into ∼30 slices, diced, and prepared for mass spectrometric analyses as described (26). Briefly, the samples were destained for 10 min in 200 μl of 50% (v/v) acetonitrile in 50 mm ammonium bicarbonate, dehydrated in 100% acetonitrile, and digested with 12.5 ng/μl sequencing grade modified trypsin (Promega, Madison, WI). The resulting peptides were bound to reverse phase resin (POROS 20 R2, Applied Biosystems, Foster City, CA), collected on a ZipTip with C18 resin (Millipore, Billerica, MA), washed twice with 20 μl of 0.1% (v/v) TFA, and eluted with 25 mg/ml 2,5-dihydroxybenzoic acid in 70% (v/v) acetonitrile and 0.1% (v/v) TFA directly onto a magnetic MALDI target (27). Mass spectrometric analyses were performed on a MALDI LTQ Orbitrap XL mass spectrometer (Thermo Electron, Bremen, Germany) (28) as described (26). Instrument calibration was performed by external calibration using standard peptide mixtures for normal and high mass ranges (ProteoMassTM calibration kit, Sigma) as described (26). The instrument was operated in positive mode, and the laser energy set to 15–20 μJ. MALDI LTQ Orbitrap MS analyses were acquired for a mass range of m/z 700–4000 with the following parameters: resolution setting, 60,000 at m/z 400; automated spectrum filter off; 50 scans/step; automated gain control on; allowing storage of 5e5 ions. MS/MS analyses were carried out by MALDI ion trap CID as described (26) using precursor ion isolation in the linear ion trap, a 2-mass unit isolation width, a normalized collision energy of 33%, an activation q of 0.25, and an activation time of 30–300 ms. The resulting MS and MS/MS spectra were inspected and processed using Qual Browser (version 2.0.7, Thermo Fisher Scientific). For the MS data, the list of singly charged monoisotopic masses was generated using Xtract within Qual Browser (XCalibur, version 2.0.7) with the following parameters: MH+, m/z 700–4000 mass range; resolution, 60,000 at m/z 400; and signal-to-noise threshold of peak picking, 2. The lists of putative proteins were obtained by database searching against the National Center for Biotechnology Information nonredundant protein database, version October 16, 2006, using the computer algorithm XProteo. The limited number of co-isolated proteins allowed us to manually inspect the MS/MS spectra and confirm the peptide sequences of the candidate proteins via assessment of the b and y ions and preferential fragmentations at the carboxyl terminus of Asp and Glu or amino terminus of Pro residues (29–32). Search parameters for MS data were: species, viruses (346,953 sequences) and Homo sapiens (152,010 sequences); protein mass, 0–500 kDa; protein pI, 1–14; mixture search, auto; number of candidates displayed, 50; enzyme, trypsin; miscleavages, 1; mass type, monoisotopic; charge state, MH+; mass tolerance, 5 ppm; fixed modification, carbamidomethylation of Cys; and variable modification, oxidation of Met. Additional parameters used for database searching using MS/MS data were: precursor tolerance, 5 ppm; fragment tolerance, 0.5 Da; and instrument: MALDI_I_TRAP. The proteins isolated with UL99 and UL32, their gi numbers, number of peptides detected, protein sequence coverage (%), XProteo scores (d′) following database searching, and the peptide sequences confirmed by MS/MS analyses are given in supplemental Tables S1 and S2. Peptide masses corresponding to trypsin autolysis were excluded. Only the proteins confirmed via the fragmentation of at least two peptides are included in the tables. The XProteo probability score indicates the discriminability (d′) for each candidate protein as a normalized distance between the distribution of the candidate protein score and that of randomly matched proteins (in units of standard deviation) based on an updated version of the ProFound Bayesian algorithm (33). A score of d′ = 4 represents a true positive rate of 0.99 and a false positive rate of 0.05.
Immunofluorescence Microscopy
Fibroblasts were plated on coverslips in 6-well plates. Cells were infected with HCMV at 0.1 pfu/cell. At the indicated times postinfection, cells were washed in PBS, fixed for 15 min in 2% paraformaldehyde, washed again in PBS, and then permeabilized for 15 min in 0.1% Triton X-100. After washing with PBS-T (PBS with 0.2% Tween 20), the cells were incubated for 1 h in PBS-T containing 2% bovine serum albumin. Cells were stained with either a mouse or rabbit primary antibody in PBS-T with 2% bovine serum albumin for 1 h at room temperature. After further washing with PBS-T, slides were incubated for 1 h at room temperature with either goat anti-mouse or goat-anti rabbit secondary antibody conjugated to Alexa Fluor 546 or 633 (Molecular Probes). 4′,6′-Diamidino-2-phenylindole dihydrochloride (DAPI; Molecular Probes) was added with the secondary antibody to visualize the nucleus. The following antibodies were used in this study: anti-pUL99 (11), anti-gB (34), anti-UL48 (35), anti-ubiquitin (Santa Cruz Biotechnology, Inc.), anti-Tsg101 (GeneTex), anti-Hrs1 (Santa Cruz Biotechnology, Inc.), and anti-TGN46 (Abcam). Cells were washed with PBS-T, mounted with Slow Fade (Molecular Probes), and viewed using a Zeiss LSM510 laser scanning microscope.
Targeted Proteomics Approach for Studying Virus-Host Associations
We have previously described a strategy that utilizes a GFP tag to both visualize the dynamic localization of viral proteins and isolate virus-host protein complexes at different times of infection. We successfully incorporated this approach for the study of cellular processes occurring during infections with Sindbis virus (36) and HCMV (22). To gain insights into processes occurring during HCMV virion assembly, we generated two viruses, BADinUL99GFP and BADinUL32GFP, containing the GFP coding region fused to the carboxyl terminus of the pUL99 and pUL32, respectively (Fig. 1A). Using this approach, the GFP-tagged proteins are expressed under the control of their native promoters, thereby ensuring that they are expressed at wild-type levels and with kinetics identical to that of the native protein.
Construction and characterization of GFP-tagged HCMV recombinant viruses. A, schematic depiction of the UL99 and UL32 genomic loci of HCMV. GFP was fused to the carboxyl termini of the UL99 and UL32 ORFs to generate the BADinUL99GFP (left panel) and BADinUL32GFP (right panel) strains. B, growth kinetics and viral yield were measured over multiple rounds of viral replication for BADinUL99GFP, BADinUL32GFP, and parental BADwt. Fibroblasts were infected at a multiplicity of 0.1 pfu/cell. Supernatants were harvested at the indicated times, and virus in the supernatants was quantified by the median tissue culture infective dose (TCID50) method. Error bars indicate standard deviations from two experiments titered in duplicate. C, fibroblasts were infected with either the parental strain, BADinUL99GFP, or BADinUL32GFP. At 72 hpi, the localization of pUL99 and pUL32 was determined by immunofluorescence using either antibodies specific for the viral proteins or, in the case of the GFP fusion viruses, the fluorescent signal of the tagged viral protein. Nuclear DNA is stained blue with DAPI. WT, wild type.
Disruption of either the UL99 or UL32 ORF results in a nonviable virus (14–16, 21). Therefore, it was critical to confirm that inclusion of the GFP tag did not affect the growth rate of these strains. Transfection of the BAC DNAs for BADinUL99GFP or BADinUL32GFP into fibroblasts revealed that both viruses were capable of replicating. The minimal impact of the GFP epitope on the growth of these viruses was confirmed by analyzing virus growth over multiple rounds of infection (Fig. 1B). Both viruses replicated with equivalent kinetics and to similar levels as the wild-type strain, confirming that the inclusion of the epitope tag did not disrupt the essential functions of either pUL99 or pUL32 in HCMV assembly.
As an additional confirmation that the GFP epitope did not interfere with the role of the viral proteins during HCMV assembly, we compared the localization of the GFP-tagged and native forms of these proteins during HCMV infection. Using monoclonal antibodies specific for pUL99 and pUL32, we confirmed that the tagged and untagged versions of the viral proteins displayed similar patterns of localization (Fig. 1C). Namely, pUL99 was found exclusively in the cytoplasm, consistent with previous results, whereas pUL32 was found in both the cytoplasm and nucleus during HCMV infection.
Upon confirmation that BADinUL99GFP and BADinUL32GFP behaved like wild-type viruses, we next identified protein binding partners for these two proteins at 72 hpi, a time that marks an early stage of the viral assembly process. We chose this time based upon the observation that pUL99 and pUL32 localize to separate compartments at this time but merge into a single compartment at later times (see below). Fibroblasts were infected with BADinUL99GFP or BADinUL32GFP, cells were harvested 72 h later, and GFP-containing protein complexes were isolated on magnetic beads covalently coupled to anti-GFP antibody. Following extensive washing of the captured complexes, proteins were resolved by gel electrophoresis, the entire gel lanes were sliced, and the proteins in each gel slice were subjected to enzymatic digestion and analyzed by mass spectrometry. Figs. 2 and 3 indicate the proteins isolated with pUL99 and pUL32, respectively. A complete list of the identified proteins, sequence coverage, number of peptides, scores derived from database searching, and peptides confirmed by MS/MS analyses can be found in supplemental Tables S1 and S2.
pUL99-interacting partners at 72 hpi. A, UL99GFP-interacting proteins were isolated by affinity purification, resolved by gel electrophoresis, and identified by sequential MS and MS/MS analyses. *, peptides corresponding to ubiquitin (UBQ) were detected between 50- and 75-kDa molecular mass markers, and therefore its exact location could not be determined. B, representative MALDI LTQ Orbitrap CID spectra of [M + H]+ ions of peptides corresponding to ubiquitin. Following CID, the generated fragment ions were detected at the linear trap level.
pUL32 associates with clathrin and components of viral tegument at 72 hpi. A, UL32-GFP-interacting proteins were isolated via affinity purifications, separated by gel electrophoresis, and identified by mass spectrometry. B, representative MALDI LTQ Orbitrap CID spectra of [M + H]+ ions of peptides corresponding to clathrin (CHC), obtained from the region indicated in A. Following CID, the generated fragment ions were detected at the linear trap level.
pUL99 and pUL32 were not isolated with each other in agreement with their distinct localization at 72 hpi (see Fig. 6). The majority of the proteins isolated with pUL99 and pUL32 are coded by the virus and have previously been shown to be components of the HCMV virion (37). Components of all three elements of the virion (nucleocapsid, tegument, and envelope) were identified in the isolations. This confirmed that the lysis conditions used were suitable for extraction of both lipid-embedded proteins and nucleocapsids. We have not yet confirmed the large number of interactions among virus proteins predicted by this analysis.
pUL99 Colocalizes with Components of ESCRT Pathway
Several cellular proteins were identified in the pUL99 immunoisolation, including peptides corresponding to ubiquitin. Although ubiquitination of proteins is often associated with degradation, ubiquitin modification is also associated with specific trafficking events. Ubiquitinated proteins are sorted and trafficked by the ESCRT pathway (38–40). Ubiquitin-binding proteins in this pathway bind to and initiate a set of vesicular transport events that coordinate traffic from the endoplasmic reticulum/Golgi with components of the endosomal network. Elements of the ESCRT pathway have recently been shown to be important for the assembly of HCMV virions (8). Together these data led us to hypothesize that pUL99 traffics through the ESCRT pathway.
To test whether pUL99 trafficking utilizes the ESCRT pathway, we compared its localization with two ubiquitin-binding proteins of the pathway, Tsg101 and Hrs. Tsg101 is a member of the ESCRT-I complex, and it is the mammalian homologue of the vacuolar protein sorting protein Vps23, which together with Vps28 and Vps27 forms the ESCRT-I protein complex in Saccaromyces cerevisiae. Hrs is the mammalian homologue of Vps27 and is known to be required in the sorting of proteins in the multivesicular body pathway. Both proteins have been implicated in the selection of cargo proteins into the ESCRT pathway by virtue of their ubiquitin binding domains (41, 42). At 72 hpi, pUL99 localized with cytoplasmic ubiquitin in the viral assembly zone during HCMV infection, and we observed substantial overlap in the localization of pUL99 with both Tsg101 and Hrs (Fig. 4A). Furthermore, our analyses of virions by Western blot using an antibody specific to ubiquitin indicated the presence of several putative ubiquitinated proteins (Fig. 4B), including a prominent band consistent with the molecular mass of the posttranslationally modified pUL99 at ∼28 kDa. The absence of Tsg101 and Hrs in our mass spectrometric analyses of pUL99 interactions may be a result of the stringent conditions that we had to utilize to be able to isolate pUL99 from vesicles. Indeed, Tsg101 and Hrs are known to bind ubiquitin via multiple low affinity binding domains. Although determining the identity of the viral proteins that are ubiquitinated requires further study, our findings nevertheless suggest that pUL99 might utilize the ESCRT pathway to facilitate trafficking of viral proteins through the assembly process and provide a potential molecular mechanism behind the observation that the ESCRT pathway is important for HCMV virion assembly (8).
HCMV pUL99 colocalizes with ubiquitin-binding proteins of ESCRT pathway. A, at 72 hpi, UL99-GFP colocalized with ubiquitin (UBQ), Tsg101, Hrs1, and TGN46. Nuclear DNA is stained blue with DAPI. TGN46 is a cellular marker of the trans-Golgi complex, previously shown to colocalize with pUL99 during viral assembly. B, Western blot analysis of virion preparations indicating the presence of ubiquitinated proteins at ∼28, 50, and 160 kDa. M, medium alone; 5K, 25K, and 50K, the number (×1000) of infectious virions loaded in each lane.
pUL32 and pUL99 Traffic in Different Pathways
Our analysis pointed to several new interactions between pUL32 and cellular proteins. Of particular interest is the apparent association with the clathrin heavy chain (Fig. 3). Clathrin-coated vesicles originate in multiple locations in the cell, and their formation has long been associated with vesicular trafficking (43). The point of origin of clathrin-coated vesicles can be determined by using the drug brefeldin A (44). Brefeldin A disrupts clathrin-coated vesicle formation from Golgi, whereas it has no effect on clathrin-coated vesicles formed elsewhere in the cell. Clathrin has been found to be required for late events in the HCMV life cycle (45, 46) as treatment with brefeldin A inhibits envelopment. In addition, clathrin is found in highly purified extracellular virions (37). Together these results suggested that HCMV virion assembly involves clathrin-coated vesicle formation from the endoplasmic reticulum or Golgi. We therefore hypothesized that the pUL32-clathrin interaction was important for viral assembly.
We treated HCMV-infected cells with brefeldin A to assess the impact of inhibition of clathrin-mediated trafficking on pUL99 and pUL32 localization. Treatment with brefeldin A did not result in a significant change in the localization of pUL32, consistent with previous results (6), but it markedly altered the localization of pUL99 (Fig. 5). The protein remained near the nucleus, but it was limited to a more compact domain in the presence of drug. This experiment also confirmed that the two proteins localize quite differently at 72 hpi. These results demonstrate that pUL99 and pUL32 traffic independently of one another during the initial stages of virus assembly: pUL99 utilizes a brefeldin-sensitive pathway, and pUL32 travels a drug-insensitive path. Rather than progressing directly from the nucleus to the assembly zone where pUL99 is located, pUL32 traffics through a clathrin-associated, brefeldin-insensitive pathway.
Brefeldin A sensitivity defines pUL99 and pUL32 as residing in separate vesicles at 72 hpi. Fibroblasts were infected with either BADinUL99GFP or BADinUL32GFP. 3 h prior to fixation, cells were treated with brefeldin A (BFA; 3 μg/ml) and fixed, and localization of the pUL99 and pUL32 proteins was determined. Nuclear DNA is stained blue with DAPI.
Nucleocapsids and Tegument Constituents Traffic with pUL32
Examination of the viral proteins predicted to be associated with pUL32 provided additional insight into the process of viral assembly. All of the components of the HCMV nucleocapsid were present among the predicted pUL32 binding partners (supplemental Table S2). This is not surprising given previous reports describing the interaction of pUL32 with the HCMV major capsid protein (18). In addition to major capsid protein, we identified the capsid proteins pUL46, pUL85, and UL80.5, arguing that pUL32 traffics with nucleocapsids. These proteins were not identified in the pUL99 immunoisolation (supplemental Table S1). Numerous tegument proteins (e.g. UL48, TRS1, UL69, UL97, UL25, and US22) were also isolated with pUL32. Coupled with the finding that pUL99 and pUL32 traffic independently through the cytoplasm during the early stages of virion assembly, these results define two independent trafficking events required for the assembly of HCMV virions.
gB Initially Traffics in a Third Compartment, Separate from pUL99 and pUL32
Although we recovered numerous glycoproteins in our isolations, the major viral glycoprotein gB was not detected. This might reflect the fact that isolation of the membrane glycoprotein was impeded by solubility issues. Alternatively, gB may reside in a separate location from the pUL99 and pUL32 compartments described above. To distinguish between these possibilities, we determined the localization of gB in comparison with pUL99 and pUL32 at 72 hpi. In a control experiment, pUL99 and pUL32 were again found to reside in different compartments (Fig. 6, top panels). Although gB exhibited partial colocalization with both pUL99 (Fig. 6, middle panels) and pUL32 (Fig. 6, bottom panels), the vast majority of the gB was localized in distinct small puncta throughout the cytoplasm. These images argue that gB, pUL99, and pUL32 begin the process of viral assembly in three distinct cytoplasmic compartments.
HCMV pUL99, pUL32, and gB reside in separate compartments at early stages of viral assembly. Primary human fibroblasts were infected with the indicated virus. 72 h later, cells were fixed and stained with the indicated antibodies. Nuclear DNA is stained blue with DAPI.
Spatial and Temporal Regulation of Virion Assembly
The finding that three virion proteins reside primarily in separate locations in the cytoplasm at the early stages of the assembly process suggested that these domains likely merge to form a functional assembly compartment. Indeed, at 96 and 120 hpi, we observed numerous instances of vesicles containing either pUL99 plus pUL32 (Fig. 7, top panels) or pUL99 plus gB (Fig. 7, bottom panels). pUL32, pUL99, and gB were found to colocalize along the periphery of these large vesicles, and structures positive for single antigens were often found in the lumen of some large vesicles. In particular, we observed numerous intralumenal structures containing pUL32 (Fig. 7, top panel, arrow). The exact nature of these structures remains unknown.
pUL32-, pUL99-, and gB-containing compartments merge as infection progresses. Infected fibroblasts were assayed for the indicated proteins at 96 (top panels) or 120 hpi (bottom panels). Nuclear DNA is stained blue with DAPI. The white arrow points to an example of pUL99 and pUL32 vesicles in the process of merging.
pUL99 and pUL32 Associations at 72 hpi
We present here, to our knowledge, the first targeted proteomics study of virus-host protein interactions occurring during HCMV virion maturation. We focused our analyses on pUL99 and pUL32, two viral proteins known to be essential to the virion assembly process. The use of GFP-tagged pUL99 and pUL32 in the context of the viral genome allowed us to integrate information regarding their localizations and interactions during the progression of virion assembly. Using cryogenic cell lysis, affinity purifications on magnetic beads conjugated with anti-GFP antibodies, and mass spectrometry, we identified numerous novel interactions of pUL99 and pUL32 (Figs. 2 and 3). These analyses also confirmed the previously reported interaction of pUL32 with major capsid protein (18). The co-isolations of these virus and host proteins are not definitive proof of direct interactions as these associations may be the result of indirect as well as some potential nonspecific interactions. Noteworthy, the majority of the proteins identified in the isolates of pUL99 and pUL32 are known to be present in cell-free virions (37), representing components of the envelope, tegument, and capsid. In agreement with our observation of the distinct localization of pUL99 and pUL32 at 72 hpi (Figs. 5 and 6), the two proteins were not isolated with each other. Furthermore, the overlap between the viral proteins isolated with pUL99 and pUL32 was limited. In addition to providing temporal interaction maps, these studies also served as a demonstration of the expression of certain viral genes that have not been previously reported, such as RL12.
Temporal Regulation of Intermediate Virion Assemblies
Altogether, the observed associations led to insights into intermediate assemblies of tegument, envelope, and capsid components prior to forming the mature virions (Fig. 8). In particular, the presence of two host proteins, ubiquitin and clathrin in isolates of pUL99 and pUL32, respectively, led us to probe for the presence of parallel distinct processes. We demonstrated that at 72 hpi pUL99 colocalized with ubiquitin as well as Hrs and Tsg101 (Fig. 4A), two ubiquitin-binding proteins of the ESCRT pathway. This observation is in agreement with previous reports demonstrating that the ESCRT pathway is utilized by many RNA (e.g. human immunodeficiency virus, type 1 (47)) and several DNA viruses (including HCMV (8), herpes simplex virus 1 (48), and likely HHV6 (49)) during the envelopment process. Our results indicated the presence of another distinct process occurring in parallel at 72 hpi as pUL32 associated with clathrin and localized in cytoplasmic compartments insensitive to brefeldin A treatment around the periphery of the viral assembly zone. As a third differentially localized process at 72 hpi, the major viral glycoprotein gB localized predominantly to another distinct compartment. These results led us to propose that at least three distinct parallel processes occur at 72 hpi: (i) ubiquitin-mediated ESCRT trafficking of pUL99, (ii) pUL32 trafficking through clathrin-associated vesicles from a non-Golgi source, and (iii) gB trafficking in yet a third compartment (Fig. 8). These distinct compartments proceed to merge as the infection progresses as visualized at 96 and 120 hpi (Figs. 7 and 8), concomitant with an increase in the resulting vesicle size (>2 μm at 120 hpi). These results represent the first observation that the final virion membrane may be derived from the fusion of these three distinct vesicles into a single membrane. The resulting large vesicle may represent the final site of virion assembly.
Proposed model of trafficking events involved in HCMV virion assembly.
These results initiate numerous interesting follow-up questions. What drives the fusion of these three vesicles? Regulation of vesicular traffic in the cell is a complex process, involving coat proteins, GTPases, and proteins that regulate vesicular fusion (e.g. soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins) (50). It is unclear which molecular mechanisms are utilized to direct the transport or fusion of vesicles involved in HCMV virion maturation. The differential sensitivity to brefeldin A indicates that distinct types of vesicular transport are involved in virion assembly. However, additional experiments targeted at identifying vesicular components that direct these events are necessary for a complete understanding of these processes. Another question raised by these findings is whether these parallel trafficking events are essential for the production of the mature virion. Although the three observed vesicles may be necessary for the maturation of the virion, these processes may also represent co-evolved alternative mechanisms. A detailed understanding of HCMV virion assembly clearly requires further work on other HCMV virion proteins and associated host factors. This study provides important insights into virion assembly and suggests the existence of parallel processes occurring during virion maturation.
We thank members of the Cristea and Shenk laboratories for helpful advice, comments, and criticisms. We also thank Joe Goodhouse for assistance with microscopy (Microscopy Core Facility at Princeton University). We also thank the many scientists in both the HCMV and proteomics field upon whose work this study builds.
↵§ Supported by American Cancer Society Postdoctoral Fellowship PF-07073-01-MBC.
↵* This work was supported, in whole or in part, by National Institutes of Health Grant DP1DA026192 from the National Institute on Drug Abuse (to I. M. C.) and Grants CA85786 and CA82396 (to T. S.). This work was also supported by Princeton University start-up funding (to I. M. C.) and an American Cancer Society Postdoctoral Fellowship PF-07073-01-MBC (to N. J. M.).
This article contains supplemental Tables S1 and S2.
↵1 The abbreviations used are:
HCMV
human cytomegalovirus
ESCRT
endosomal sorting complex required for transport
green fluorescence protein
bacterial artificial chromosome
plaque-forming unit(s)
hours postinfection
Bis-Tris
2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol
LTQ
linear trap quadrupole
4′,6′-diamidino-2-phenylindole dihydrochloride
immunoglobulin heavy chain binding protein
glycoprotein B.
Received October 16, 2009.
Revision received December 16, 2009.
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc.
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You are going to email the following A Targeted Spatial-Temporal Proteomics Approach Implicates Multiple Cellular Trafficking Pathways in Human Cytomegalovirus Virion Maturation
Nathaniel J. Moorman, Ronit Sharon-Friling, Thomas Shenk, Ileana M. Cristea
Molecular & Cellular Proteomics May 1, 2010, First published on December 20, 2009, 9 (5) 851-860; DOI: 10.1074/mcp.M900485-MCP200
Vol. 9, Issue 5


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Top Music Apps for iOS in India
HomeAll Posts...Top Music Apps for iOS in India
Apple Blog iPhone
July 15, 2017 by suneil
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Founded back in 2007, Saavn has been one of the first apps to bring Indian music across different genres Saavn provides unlimited free access to music content across all genres and all major regional languages.
This application has a lot of streaming features including setting the speed of streaming. You could even choose the radio station of your favourite artist and listen to songs that have been exclusively sung by them Through savan you will be able to find the latest tunes and some exclusive for Saavn content here. The app even provides playlists categorised by genre, artist, or your mood. There's also a wide selection of podcasts, radio channels based on drama, mystery, comedy, and sports.
Saavn can also be used as a standalone player for your device as it reads the music data on your device with ease. Just press the 'On my phone' option under Saavn menu and you can access tracks and playlists from your device for offline play
If you subscribe to the pro version of the app (Saavn Pro), you could get access to unlimited storage space for downloaded songs on up to five devices. Saavn is available on iiOS for as low as Rs 99/month.
Bas Bajna Chahiye Gaana' is a tune that has caught the attention of several TV audience and mobile device users as the Gaana application is being aggressively advertised on TV , Gaana offers you unlimited, free access to all your favourite tunes, Radio Mirchi, Bollywood music, and even music videos
Gaana's UI look and feel carries a very relaxed approach with red and white theme running through the entire app. It features curated playlists from experts and other users. Gaana contains a huge collection of Bollywood, International, regional content such as Marathi, Bengali, Rajasthani, Bhojpuri, Malayalam etc.
You can subscribe to Gaana Plus (paid subscription) for Rs 297 for 3 months , 549 for 6 months and 999 for 1 year.
Wynk
Developed by Airtel, but is available for all users. Wynk currently hosts up to 2.6 million songs across Indian and International music. It ensures high quality music streaming for free on demand. Wynk covers all genres including Bollywood, Pop, Rock, Metal, Bhangra, Devotional, Romantic, party songs etc. It also moves into regional songs from Marathi, Bhojpuri, Rajasthani, Bengali etc. languages. This app contains a curated music library along with multiple playlists based on moods and genres. You could even set a caller ring back tone (caller tune) using just about any tune, specifically if you are an existing Airtel customer. This music app is best known for easy search or discovery of songs
Wynk supports unlimited ad-free streaming. It offers a multi-lingual user interface with which you can interact in English or Hindi. You get access to internet radio that enables you to listen to your preferred genre of music without any interruptions. A major USP of Wynk is that it allows you to select the quality of streaming and sound.
Wynk provides a first month free trail for all new users with ad-free music streaming and download. Later you can opt for monthly subscription of Rs 99 for non-Airtel Android users, while Airtel users get the same for as low as Rs 29. Airtel users get Free Wynk subscriptions on certain Data recharges. Wynk is available on iOS.
Humgama
Hungama is another such music app favourable for Indian users with over 3.5 million songs and also videos of Bollywood, Regional and International flavors. It's adaptive streaming feature guarantees a seamless music experience and works without buffering on slow network speeds. You can listen to celebrity curated playlists and radio channels. You can watch music videos and also view lyrics to setup a mini karaoke experience. Unlike Saavn and Gaana, this application automatically downloads songs that you have played to enable their offline use as well.
The app layout is simple and lets you browse through content without any difficulty. It has a shiny looking music player but is clean and offers very good readability throughout the app. It has a feature onboard called 'Discovery' where you can set your mood and tempo of the song to discover random music content as per your requirement
Hungama features a reward system by which you can earn coins by inviting friends, sharing and creating playlists on the app. These points can be redeemed later for subscriptions, movie tickets, discount vouchers, merchandise and more.
Music, video and radio streaming is free but with ads. Subscriptions start at Rs 99.
We feel Wynk s amalmagation of a huge library of Indian and International music and well curated playlist, combined with a low price and ease of discovery make it a very appealing proposition to the Indian market. Wynk can't be used on the web, unless you are using a mobile phone, but most people are going to be accessing it from mobile phones, so its not going to be much of a problem.


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Cremation and Modern Jewish History
Posted on July 27, 2011 by Alan Brill | 18 Comments
With all the buzz about Amy Winehouse, I am surprised that none of the recent articles have presented the history of the issue. Most of the newspapers are simply quoting chabad.org. I don't usually give simple bibliographies for a blog post but in this case it may be needed. (Nothing in this post is intended to decide any legal issues.)
In 1873, an Italian inventor presented in Vienna his new machine for cremation. It ushered in an era when cremation was seen as more modern, scientific, and hygienic than burial. It was seen as dignified and respectable. It sweep the Protestant world for several decades. They said that if God can resurrect the dead from bones, then he can just as easily do it from ashes. However, cremation was banned by the Catholic Church until 1963.
Rabbi Elijah ben Amozegh, Chief Rabbi of Livorno, wrote a treatise on the subject Ya'aneh Ba'esh (Livorno,1886), and called a conference of the Rabbis who banned cremation. Yet, Moses Israel Tedeschi, rabbi of Trieste, published a responsum in 1890 in which he not only tried to prove that cremation was not opposed to the spirit of Judaism, but asked that at his death his own body should be disposed of in this way. Cremation was avoided but would not write someone out of the fold.
For more on Italy, see David Malkiel, "Technology and Culture Regarding Cremation: A Historical and Phenomenological Analysis," (Hebrew) Italia 10 (1993) pp. 37-70.
Similar to Italy, England Chief Rabbi Nathan Marcus Adler, who held office from 1845-1890: "I beg to state that whilst there does not exist any precept prohibiting the interment in a Jewish cemetery of the ashes of a person who has already been cremated, our law is decidedly and emphatically opposed to the practice of cremation."
Chief Rabbi Herman Adler, Nathan Marcus Adler's son and his successor in office, stated: "There does not exist any precept prohibiting the interment in a Jewish cemetery of the ashes of a person who has already been cremated, an opinion supported by other eminent rabbis. We accordingly permit such a burial. At the same time we earnestly beg you not to construe this permission into a sanction of the practice of cremation."
And therefore after the fact the practice was allowed full burial service by Burial Society of the United Synagogue of London: "The society shall not make any arrangement whatever for cremation. Where cremation is nevertheless to take place a service may be held at the house prior to the removal of the body, and if the ashes be encoffined then interment may take place at a Cemetery of the United Synagogue and the burial service shall be conducted there at the time of the interment." (They have subsequently revoked such after the fact tolerance, but still tolerated by the Liberal movement.)
But in Germany things were different. In 1905, Dr. Meir Lerner of Altona, Germany, published his famous Hayyei Olam (Berlin 1905), containing answers of Rabbis from every country in the world that had a Jewish community with a rabbi. Not only did they all condemn the practice, but also, with a few notable exceptions, they prohibited doing anything with the ashes, such as burial in a Jewish cemetery, or for the survivors to observe the laws of mourning.
Adam Ferziger (Bar Ilan) has been giving an academic paper in the making for several years: "The Hamburg Cremation Controversy: early 20th century Orthodoxy and the boundaries of Jewish identity." In the paper, he explained how this became a major cause célèbre and ideological battle culminating in considering those who are cremated as not deserving of shiva.
When I heard the paper in a room of historians of modern Judaism, the question was: why the extreme reaction in Germany and not in Italy or England. The basic consensus was that in German Jewry everything rose to dramatic ideological battle lines and identity proportions of inclusion and exclusion, which was not true about other countries.
Israel was the most categorical and did not have cremation until 2005. In 1935, the Rabbis of Jerusalem issued this ban against cremation: "We hereby notify all our fellow Jews that every Hevra Kadisha may not handle the ashes of those cremated, and they may not receive the ashes to bury them in a Jewish cemetery" 10 Tevet 5695 (1935)… (Responsa Da'at Kohen of Rabbi Avraham Yitzhak Kook,382).
In a more recent paper Adam Ferzinger, "Ashes to Outcasts: Cremation and Jewish Identity Before and After the Holocaust." Points out the change in rhetoric to now eschew cremation, even among secular Jews, because of the Holocaust. A 1990 Reform responsum notes: "Reform Jewish practice permits cremation… although… we would, after the Holocaust, generally discourage it because of the tragic overtones."
In the 19th century Rabbinic statements from Rabbi Yitzhak Elchanan and reiterated by Rabbi Chaim Ozer, cremation is an implied statement of rejection of the concept of resurrection.The reason at the end of the 19th century for not cremating was still becuase of the need to be whole at the time of resurection of the body. In the 20th century rabbis explain the avoidance as a to a reason based on human dignity. We witness a change from rejecting cremation in the 19th century because of the need for a complete body for resurrection to Meir Lerner's 1905 collection of Talmudic sources of human dignity and the prohibition to harm a body even after death. The responsa of Rabbi David Zvi Hoffmann as well as the one written by Rabbi Abraham Isaac Kook frame the issue as issues of human dignity and not to harm the body of the deceased. It is now seen as forbidden to destroy or mutilate a human body, even after death.
Jon D Levenson at the start of his work Resurrection and the Restoration of Israel: The Ultimate Victory of the God of Life. (Yale University Press, 2006) describes the loss of a sense of resurrection of the body among modern Jews. This shift away from resurrection exemplifies what the historian Michelle Vovelle documents as the cultural shift away from death by the end of the 19th century. For him, it is a removal of relgion and theology from our lives.
Now in the 21st century, we have a turn to cremation in American non-Jewish culture because it seems more clean and final, more economical, as well as more "Buddhist." More than half of Americans in Western states are being cremated. It will increase in the Jewish community, so be prepared to deal with the issue and even create new traditions.
For some sources:
Jewish Encyclopedia
Rabbi Raymond Apple
Does Jewish Law Permit Cremation? Rabbi Isaac Klein edited by Rabbi David Golinkin
Reform Movement- CCJR Responsa
Chabad Answer with a stronger emphasis on resurrection and the soul
For those who will be quoting this in their classes, please give full credit to the hard work of all the scholars cited and to this blog.
Postscript Update- I received an email from someone who claims that one of the YU Roshei Yeshiva told his aveilut shiur that, off the record, he knew of nothing halakhically wrong with cremation, though there are kabbalistic reservations.
This entry was posted in Uncategorized and tagged cremation halakhah, cremation judaism, cremation sitting shiva, jewish cremation. Bookmark the permalink.
18 responses to "Cremation and Modern Jewish History"
Rabbi Yaakov Feldman | July 27, 2011 at 7:21 pm | Reply
Thank you for this. I hope to dive into the material at a better time. My experience is many Jews do it because it's far cheaper than ground burial rather than due to any Buddhist link.
Moshe Shoshan | July 28, 2011 at 6:52 am | Reply
Could you please flesh out the halachic arguments a little. Isnt this clearly nivul hamet?
AS | July 28, 2011 at 9:17 am | Reply
Moshe,
Jewish burial rites tend to favor practices that lead to the rapid decomposition of the body. Arguably cremation is simply an accelerated process and not intended to mutilate the corpse.
I hope that "fleshes it out" for you – or was that not an intended pun?
HS | July 28, 2011 at 6:53 am | Reply
Agreed with Rabbi Feldman. Space is limited, and expending millions of acres on cemeteries will become increasingly expensive, and make it increasingly difficult for survivors to visit. The churchyards of England and the multi-layered catacombs of Italy attest to the long-term impracticality of whole-body burial.
IH | July 28, 2011 at 2:31 pm | Reply
Cremation is the societal norm in the UK (~70% cremation vs. ~30% burial) and this appears to have penetrated non-Orthodox Judaism. As an example, see the membership package for Jews wishing to join the Belsize Square Synagogue in London. It is a successful Independent shul, founded by German refugees (click on Burial and Cremation Scheme for the details). http://tinyurl.com/3p2388k
Arie Folger | July 29, 2011 at 7:29 am | Reply
Actually, if you think this is going to catch on among more Jews, we be quite behind the curve, because cremation is now becoming uncool, as it is not environmentally sound. Fancy caskets are similarly slowly falling out of grace.
I blogged on this here: The Ecologically Correct Funeral.
Of course, such trends are notoriously difficult to forecast.
Adam Ferziger | July 29, 2011 at 7:36 am | Reply
Dear Alan,
Thank you for bringing this issue and my work on it to the attention of your readership. One caveat, to the consternation of Rabbi Lerner quite a few German rabbis permitted burial of the ashes. They required, however, that they be placed in a separate section of the burial ground.
An article that I wrote addressing the German Orthodox context of the dispute is set to appear in the upcoming volume of the Leo Baeck Institute Year Book. A second one that focuses on the significance of the debate from the perspective of history of halakhah, particularly approaches to nonobservance, is under review for publication in an additional journal.
Since the first oral presentation, my interest and appreciation for the richness of the subject of cremation and twentieth century Jewry has grown dramatically. I have gathered a great deal of relevant sources and plan to produce a full-length monograph that spans the entire period. To offer one small example, recently I shared some of my materials with a group of rabbis from across the denominations. Afterwards, one participant told me he knew of a case in the 1970s in which two congregants actually asked to be cremated and explained to the rabbi that they saw this as a way of identifying with the martyrs of the Holocaust
If you or any of your readers are aware of relevant materials (rabbinic writings of all denominations – responsa, sermons, essays; personal considerations of the issue, synagogue or cemetery statutes, memoirs, oral history), I would very much appreciate being sent copies or at least bibliographical references.
I can be reached at ksferz@gmail.com.
Adam Ferziger
Alan Brill | July 29, 2011 at 8:46 am | Reply
What is the context for cremation being discussed in 1935 by the Rabbis of Jerusalem? What precipitated it?
Also what is the background behind Maurice Lamm's stating that kaddish is not said?
Dave | July 29, 2011 at 7:53 am | Reply
I understand that our ancestors in Ancient Israel used to bury the body and then after some years passed, would dig up the bones and deposit them in a "bone box". That may sound macabre, but possibly that would be halachically acceptable? Would anyone care to comment?
Dave — The Torah itself seems to countenance the transfer of bones. Jacob asked his sons, "Do not bury me in Egypt" (Bereshis 47:29). Joseph, on the other hand, asked his brothers "You must carry up my bones from here" (50:25). This seems a pretty clear request for temporary internment and later transfer. If Joseph thought it inappropriate to do that, he could have made the same request his father did.
rabbichaplain | July 29, 2011 at 8:03 am | Reply
As someone often confronted with Jewish cremation in my hospice work, I thought I would add in a couple of points.
1. I have come across an different phenomenon regarding cremation and the Holocaust. While many eschew the practice because of the cremation of our brethren by the Nazi's, there are people who specifically want to be cremated as a means of connecting with their deceased who were cremated.
2. As a hospice chaplain, the subject of cremation is a common conversation. I am personally averse to cremation as a form of burial. However, as a chaplain, whose primary role is being supportive to people through spiritual means, I do not intervene to prevent someone from making a choice I disagree with. I had a situation early in my career in which a non-orthodox family was planning to cremate their mother at her request. They had made all the arrangements, and then the patient's son comes over to me and says, my brother-in-law told me that cremation is not allowed according to Jewish law. In response, I offered him two approaches. He could either decide not to cremate his mother because cremation is not allowed (the information from your post notwithstanding, and not known by me at the time) or he could decide to follow her wishes. Either way, I was not going to abandon them when they needed support. This is a personal approach of mine, not something that I expect everyone to adhere to, but one which I believe serves the situation well.
3. On the same subject, since many Jewish people are exposed to the Jewish views on death and dying through R. Maurice Lamm's The Jewish Way of Death and Mourning, it is important to note that he categorically says it is prohibited, which for many is all they ever know. There is another source, much more nuanced, written by another Orthodox Jewish chaplain, from whom I got the impetus to distinguish between a decision I disagree with and the need to still support the grieving family. It is found in the book To Walk in God's Ways, by R. Joseph Ozarowski. But even there, he provides texts that seemingly indicate more than a mere aversion to the idea.
Lamm is most likely based on Lerner. I haven't studied the 1935 case yet.
Rabbi Regina Sandler-Phillips | July 29, 2011 at 4:08 pm | Reply
Thanks for sharing this. Yes, our historical and halakhic sources are much more nuanced than our popular consciousness on these issues.
While cremation may indeed seem "more clean and final, more economical," most people (Jews among them) are unaware of information that runs counter to this perception. In my experience, the primary unaddressed issues are vulnerability to criminal desecration (the real problem of nivul hamet), environmental costs (and hence the violation of bal tashkhit), and avoidance of the messy human need to grieve our losses.
In recent years I have been compiling "Dust and Ashes" resource materials to facilitate more effective dialogue on Judaism and cremation. These materials are now available with a 45-minute telephone orientation and tutorial which includes:
* Strategies for opening and sustaining communication about cremation and end-of-life values
* Surprising and little-understood complexities of cremation for Holocaust survivor families
* Basic but little-known facts about the cremation process
* Actual environmental costs of cremation–and environmental justice issues of crematory construction and maintenance
* Implications of the absence of sh'mirah and levayah
* The importance of direct rabbinic and lay leadership involvement in practical advocacy for kibbut hamet
* Accessible Biblical and classical rabbinic texts that highlight basic Jewish principles of kibbud hamet
* Excerpts of selected contemporary responsa (including some of those mentioned above)
Those who are interested in receiving these materials and the accompanying telephone tutorial can contact me via waysofpeace (at) earthlink (dot) net.
With many blessings for Shabbat Shalom and beyond,
Rabbi Regina Sandler-Phillips, MSW, MPH
http://isabellafreedman.org/sacredundertaking
"We sustain the poor…and visit the sick…
and bury the dead…and comfort the bereaved…
for these are ways of peace."
(Jerusalem Talmud, Tractate Gittin)
konneliyahu | July 30, 2011 at 4:45 pm | Reply
In the beginning of the Common Era when some were buried in tombs, there was the duty of opening the tomb after three days to make sure the supposed deceased were dead. You can also cross reference in the Tanakh, 2 Kings 13:21, that this indeed happened. There seems to be an aversion to being buried alive (since orthodox are not embalmed how would one know) and cremation certainly negates this. And I wouldn't be so quick to say we know death better with our advanced medicine, especially since business (medicine is business) is trying to squeeze productivity out of fewer workers and there is the golf course of course. There are still people lucky enough to escape being buried alive but I wouldn't rely on it.
Ben Elton | July 31, 2011 at 2:42 pm | Reply
The Rev. JF Stern, for many years the Minister of the East London (United) Synagogue in the late 19th and early 20th centuries, instructed in his will that he be cremated, and he was.
The United Synagogue in London has always taken the pragmatic view that Jewish remains are Jewish remains, and whatever condition they may be in is not the business of the Hevra Kadisha, unless they took care of the funeral. Interestingly, the United Synagogue Hevra Kadisha is actually called the Hevra Gemilut Hasadim.
Geoffrey Stern | August 1, 2011 at 8:59 pm | Reply
Facinating blog on cremation and Jewish Law… we need more blogs like this that question and review accepted doctine in a scholarly, historical and reasonable manner.
shueyr | August 7, 2011 at 2:28 am | Reply
Funny that nobody mentioned how King Shaul was kinda cremated…
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