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Data in Listboxes can get unruly causing the users to do a lot of scrolling. One way to rein them in is to allow the user to filter the Listbox by typing in a Textbox. It's kind of like autocomplete. In this example, a Listbox is populated with all the customer names from the Northwind database's Customers table. The user types in the Textbox and the Listbox is automatically filtered.
The Change event of the Textbox is used to filter the Listbox.
Dim i As Long
Dim sCrit As String
'Add asterisks around text for all matches
'UCase is used to make filter case-insensitive
sCrit = "*" & UCase(Me.tbxFind.Text) & "*"
With Me.lbxCustomers
'Start with a fresh list
.List = vaCustNames
'Loop through the list backward – always a good
'idea when you're deleting stuff
For i = .ListCount – 1 To 0 Step -1
'Remove the line if it doesn't match
'UCase used again here
If Not UCase(.List(i)) Like sCrit Then
.RemoveItem i
End If
Next i
End With
End Sub
I put the customer names in a variant array in the userform's Initialize event. The array variable is a module level scope so I can use it anywhere in the userform's module. It's a little brute force to refresh the list with every change, but it wasn't very slow for me and the 91 names in the list. If you had a much bigger list, you might track the length of the Textbox and only start fresh when the length goes down.
If Len(Me.tbxFind.Text) < lOldLen Then
'Start with a fresh list
.List = vaCustNames
End If
lOldLen = Len(Me.tbxFind.Text)
lOldLen is another module level variable that stores the previous length of the text in the Textbox. That may speed things up a bit with a large list.


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Gold is the most precious metal that is highly consumed by Indian consumers. People in India wear gold frequently as they believe wearing authentic designs of gold denotes their status in society. Indians also treat gold as a physical asset in which they can sell it instantly for their sudden requirements in the future.
However, the attitude of Indian people has changed drastically in terms of investment because they prefer to choose financial assets when compared to physical assets. But, in both the cases the investors would purchase the asset only when the price is low or would purchase it irrespective of price depending on their requirements.
In this blog, we'll discuss why people choose both gold and stocks for investment and also compare the benefits between them.
Gold is a physical asset with a proven track record of stable growth. Indians consider gold as a "safe haven" investment as the value of assets tends to increase when there is a depreciation in the financial market. Gold is an investment option that helps you to protect during unpredictable financial swings.
Investing in Gold is an easy option to invest. All you require is an easy liquidation with storage options available. You can also choose gold saving schemes offered by jewelers by paying monthly installments to purchase your gold jewels.
Stocks are financial asset that helps an investor to invest in the shares listed by the companies available in the stock exchanges. Choosing the stocks in a stock market would require expert guidance as you would need to conduct technical and fundamental analysis for profitable returns. Investing in stocks can fetch you higher returns when compared to gold as the stock performance depends on the company's growth.
If you are in search of an expert who can guide you to make your investment successful in stocks, then choose the guidance of 500 per crore. Backed by a decade of experience in trading, 500 per crore guides you to choose the best performing stocks in the market. To know more details, click here!
APPROACHING PRICE RISE & FALL: This is the common factor that affects the performance of both investment options. This is because both investment options have their own market factors that determine the price. Investing in stocks can fetch you high returns when compared to gold investment but at certain market conditions the stock price might depreciate and also has the possibility to regain its market position.
FOLLOW THE THUMB RULE: Invest in Gold when the price is low and hold on to the asset till the price rises. While investing in the share market, ensure to start with a low investment as it will help you to gain better experience in the stock market. While investing in both options you should always ensure that you keep a small savings by your side to protect you from swing fluctuations.
BE CAUTIOUS: If you choose to invest in gold or stocks, ensure you never invest your whole savings on a single asset as it can lead you to huge loss in the future. While investing in gold or stocks, always diversify your investment portfolio as it will help you to overcome the loss faced while investing in one asset.
When you are asked to choose between gold or stocks, choose the best one based on your priority as both options serve their own purpose. Choosing stocks would give you a chance to earn more profit similarly while choosing gold the price may depreciate based on the dollar value. Hence, analyze your requirement and choose the best investment option!
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subset: commoncrawl


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The 'fast-lane' debate resumes over Google and tiered access
What could be a "non-story story" in this morning's Wall Street Journal which some are saying strays too far from the truth, was met with a "non-denial denial" response from Google indicating it may not be too far from wrong.
The chatter this morning is about whether Google, a company whose name actually means "big," is approaching cable companies about possible partnerships that could give certain customers higher-speed, premium access to higher-bandwidth content such as YouTube and Google Apps. This after a Wall Street Journal story painted Google as two-faced, advocating so-called "net neutrality" principles in public while making exclusivity deals with carriers in private.
The WSJ story received very high placement Monday morning in almost every blog and aggregator except, interestingly enough, Google News.
Arguably, the story may not have any actual news in it. That Google and other major content providers have been considering or actively seeking partnerships that could lead to "fast-lane" access for themselves and others willing to pay for it, has been a fact in the public record for at least three years. In the spring of 2006, it became a bone of legislative contention when the then-Republican controlled Senate Commerce Committee attempted to railroad through Congress legislation that would permit national licensing for qualified broadband providers. Proponents of the measure suggested openly that carriers could partner with content providers to offer premium-tier service to customers, which they said would be necessary in order to generate the revenue to fund nationwide broadband buildouts.
But opponents argued that such deals would give carriers and content providers the means to partition the Internet into classes of accessibility that could end up being exclusionary for smaller providers and smaller carriers. It was this debate that actually seeded the phrase "net neutrality" into the common vernacular. In the end, the opponents won the day not for having won the argument, but due to the Democrats' takeover of Congress the following year.
At that time, one outspoken opponent of net partitioning was Stanford University Law School Professor Lawrence Lessig. During the Commerce Committee debate, Prof. Lessig spoke out in favor of former FCC Chairman Michael Powell's "Internet Freedoms," an effort to codify a series of four principles about Internet access and service distribution in a manner not dissimilar to the Bill of Rights. "The now openly-stated intentions of AT&T and others to introduce access- tiering to the Internet threatens to undermine application competition on the Internet. Congress should act to avoid that result," reads Prof. Lessig's written testimony before Congress (entered into the record though not spoken aloud).
"Access-tiering will create an obvious incentive among the effective duopoly that now provides broadband service to most Americans," the Professor's statement continued. "By effectively auctioning off lanes of broadband service, this form of tiering will restrict the opportunity of many to compete in providing new Internet service...The incentives in a world of access-tiering would be to auction to the highest bidders the quality of service necessary to support video service, and leave to the rest insufficient bandwidth to compete. That may benefit established companies, but it will only burden new innovators."
In the very next paragraph, Prof. Lessig goes on to say this does not make him opposed to the concept of network owners charging end customers different fees on a value scale -- essentially saying that AT&T, Comcast, and others should have the right to enable economies of scale on their networks.
But perhaps in an attempt to fill out a story that would otherwise have only consumed three paragraphs, the WSJ this morning suggested that Prof. Lessig's most recent advice, including to Pres.-Elect Obama, that network operators be permitted to charge end customers fair but scalable rates, was evidence that he had "shifted gears," perhaps as a compromise that could grease the fast track for a federal appointment.
That suggestion infuriated the Professor, who responded on his personal blog early this morning, "The article is an indirect effort to gin up a drama about a drama about an alleged shift in Obama's policies about network neutrality. What's the evidence for the shift? That Google allegedly is negotiating for faster service on some network pipes" -- a fact which, again, has been part of the public record for years.
A common tactic for political advocates, most notably on talk shows, when faced with an uncomfortable question, is to rephrase the question and then answer that one. (Comedienne Tina Fey, in her role as Alaska Gov. Sarah Palin, demonstrated that technique in a now-classic send-up of the last Vice Presidential debate.) So this morning's response to the WSJ article from Google itself skillfully attempts to invoke that method, in a way that could only spark more skepticism about a subject that may otherwise have been left effectively diffused and rendered moot by Prof. Lessig.
"Some critics have questioned whether improving Web performance through edge caching -- temporary storage of frequently accessed data on servers that are located close to end users -- violates the concept of network neutrality," reads this morning's statement from Google counsel Richard Whitt. "As I said last summer, this myth -- which unfortunately underlies a confused story in Monday's Wall Street Journal -- is based on a misunderstanding of the way in which the open Internet works."
The technique of edge caching enables systems that cache high-volume, high-traffic content to be located more closely, in the Internet geography, to the end users. The more they request the content, the more closely those caches are positioned to the user, expediting transfer time. Whitt says that Google has been talking with ISPs about being able to collocate its own servers there, in deals that would make YouTube content conceivably closer to end users, but which would otherwise not be exclusive -- and, once again, which wouldn't be news to anyone.
Nonetheless, it's almost impossible not to notice that the WSJ story did not mention edge caching at all, focusing instead on the type of "fast-lane" provisioning that was the subject of the stalled 2006 legislation.
Last September, a bill authored by Sen. Ed Markey (D - Mass.) -- an outspoken opponent of the 2006 "fast-lane" bill -- calling for ISPs to provide the government with enhanced geographic data on their broadband service, passed Congress. Now, ISPs will be required to provide information on their services at the nine-digit ZIP code level, with the likely result being a so-called "broadband map" that could reveal whether carriers and content providers have already made deals that have never been disclosed.


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The next Spider-Man movie comes out in December and it might just be the last one for a while, according to Tom Holland.
"We were all treating [No Way Home] as the end of a franchise, let's say," Holland tells Entertainment Weekly. "I think if we were lucky enough to dive into these characters again, you'd be seeing a very different version. It would no longer be the Homecoming trilogy. We would give it some time and try to build something different and tonally change the films. Whether that happens or not, I don't know. But we were definitely treating [No Way Home] like it was coming to an end, and it felt like it."
Now, this doesn't necessarily mean that Holland's take on Spider-Man is leaving the MCU. But it does appear that solo Spider-Man projects will be on pause for a little. There is certainly a chance the character could be revisited later on in his narrative, when he's less a teenage hero and more of an established Avenger. Spider-Man could also take on a bigger role in Avengers projects, or just simply show up in other movies.
Tom Holland's take on the webslinger first debuted in Captain America: Civil War, as Iron Man's protege. The character then went on to have two solo movies, Spider-Man: Homecoming and Spider-Man: Far From Home. He also got famously snapped in Avengers: Infinity War, clinging to Tony Stark and heartbreakingly saying "Mr. Stark, I don't feel so good."
Whatever the future of Peter Parker and his friends becomes, we'll find out on Dec. 17, when Spider-Man: No Way Home hits theaters.


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The Diana Award for 2021 was given to two students from Lady Shri Ram (LSR) College and Jamia Millia Islamia for their contributions to the UN Sustainable Development Goals. Devanshi Ranjan, a final-year LSR student, has been recognised for her efforts to assist impoverished youngsters, particularly female pupils, with their education during the COVID-19 pandemic.
According to a university release, Kaif Ali, a fourth-year Bachelor of Architecture (B.Arch) student at Jamia Millia Islamia, won the award for creating a sustainable housing that helps prevent coronavirus transmission. The prize, named after Diana, Princess of Wales, is the "highest distinction" a young person can get for social action or humanitarian activities. It is distributed through the same-named charity, which has the backing of both of her sons, the Duke of Cambridge and the Duke of Sussex.
"Ali designed a prefabricated sustainable structure that would not only assist prevent virus transmission but could also accommodate refugees worldwide in the future," according to the Jamia statement. His idea is currently being implemented in Lagos, Nigeria, and has been recognised by the Indian government, the Commonwealth of Nations, and the United Nations as one of the top 11 rising innovative start-ups addressing climate change, according to the statement. "I found out that I will be honoured with the Diana award on May 5," Ranjan, who has been working with NGO Ladli Foundation Trust, told PTI.
The Diana Award, which is granted to youngsters between the ages of 9 and 25, was given to 400 people around the world.
"We congratulate all of our new Diana Award honorees, who are changemakers for their generation, from the United Kingdom and around the world. We are confident that earning this honour would encourage more young people to become involved in their communities and embark on their own journey as active citizens "Tes said


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Cairo - 10 February 2022: UNICEF announced today that the short documentary film "It Takes a Village" has won the "Best Short Documentary" award at the New York Movie Awards. The film was first screened on October 18, 2021 at a special event in El Gouna International Film Festival (GFF), under the auspices of the National Council for Women (NCW) and the National Council for Childhood and Motherhood (NCCM), in cooperation with UNICEF and with the support of the United States Agency for International Development (USAID).
The short documentary film tells the story of three girls (Alaa, Samaa & Noura) from Egypt with something in common that changed their lives and will be the reason to inspire many other girls. It also shows the challenges, the dreams and hopes for all girls in Egypt. The film is directed by Ms. Reem Osama and funded by USAID.
On this occasion, Dr. Maya Morsi, President of the National Council for Women, said: "I am proud of the 'Dawwie' national initiative and of the substantial progress that Egypt has achieved in empowering girls. The Egyptian state is exerting continuous efforts in supporting girls to play an active role in their societies and contribute to creating a better future for themselves and their country. I believe that the girls of Egypt are capable of making change and achieving more success during the coming period."
From his side, Dr. Tarek Tawfik, Deputy Minister of Health and Population for Population Affairs and the Supervisor of the National Council for Childhood and Motherhood, expressed his happiness that the film "It Takes a Village" won this award. Stating that this represents a highlight towars supporting girls' educational, health and cultural rights. He also stressed on the importance of arts and the film industry in supporting societal issues; by highlighting important issues that provide greater opportunities for societal change. Tawfik also stressed on the role of NCCM in launching the joint national initiative to empower girls "Dawwie" under the auspices of the Council and in cooperation with UNICEF, which focused since its inception on supporting the rights of girls and is one of the mechanisms that contribute to creating an enabling environment that supports the rights of girls in various fields.
Leslie Reed, USAID Mission Director said: "We are pleased to be among the partners working to support 'Dawwie's' initiative interventions, including supporting the film that won the 'Best Short Documentary' award at the New York Movie Awards. The United States of America is committed, to empowering girls and ensuring their full participation in economic and social life in Egypt."
Jeremy Hopkins, UNICEF Representative in Egypt said: "We are absolutely delighted that this film has been recognized with this award. This underscores the essence of the film which is to ensure that girls voices are heard at home and all over the world. UNICEF commits to continuing our efforts to empower every girl in Egypt to enjoy her rights and to transition to adulthood with the skills she needs."
The three heroines of the film are participants of the 'Dawwie' national initiative that aims to foster an enabling environment for Egyptian girls, especially the most vulnerable, to achieve their full potential while enhancing the participation of their families and communities as well. The initiative is currently being implemented in twenty-one Egyptian governorates.
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What Can You Do To Save Your Chuckit From Destruction By Social Media?
Dog house owners are almost definitely aware of dog toys being advertised all over pet stores. This dog toy is designed to keep your canine's jaw, mouth, and teeth wholesome. Nylabone says their canine toys can be utilized for moderate chewing points that stem from boredom, destructiveness, and stress. Its versatile structure and meat flavor help control chewing bouts, so your dog won't opt for the closest household object. Plus, bristles raised throughout chewing help control plaque, decrease tartar, and cut back tooth pain. Tip: It is a good idea to observe your dog after they're chewing on this dog toy, since they might attempt to eat small parts that break off.
We now have been there, and we get how frustrating sucky dog toys could be. And eventually, the Zogoflex Hurley Tough Dog chuck it balls medium Bone Chew Toy. Made in the USA and licensed safe, the Hurley is gentler and safer than rawhide bones, and built for long chew periods.
We first saw Rhinoplay toys at a pet merchandise current last summer season. We absolutely loved their mild weight and distinctive, puncture-proof material. The corporate claims the froth is environmentally protected and nontoxic, and ensures the product. In case your canine doesn't favor it, or destroys it in a match of enthusiasm, the company will change it.
The other toy I have is the Chuckit! Kick Fetch ball. It is available in two sizes, small or large, and my canine is a reasonably large shepherd mix so I have the large one for him. This one is actually cool if you wish to play mainly kickball or soccer along with your canine. My canine loves soccer however he destroys each soccer ball that he ever touches so he simply likes to play too rough, however this thing is tremendous sturdy.
News On Clear-Cut Chuck It Methods
This lightweight dog toy can be thrown around without fear that you will break a window. It is the Good toy for indoor video games of fetch if you find yourself snowed in. Perfect for little doggies or puppies who're teething, and the cotton fibers clear tooth as they play. And, these rope toys are washable, making certain they've a pleasant long life even if they get a bit dirty alongside the way.
There's nothing worse than buying a canine toy, and inside an hour you flip your head to see the scattered remains of what you thought was a smart buy. Avoiding this case is all about realizing how your canine will interact with certain toys. As a rule, most every plush toy goes to eventually die a messy dying if left alone with a canine that likes to chew.
Like with all good chew toys, the Gorilla Chew is accessible in sizes small to large. Goughnuts have taken the identical near-indestructible rubber used in their chew rings and created a heavy-obligation tug toy for the most powerful breeds round.
Will it is advisable to retailer your canine toys chuckit large? If that's the case, you need to take into account the scale and weight of the dog chuck it toys chuckit largeto be sure that it can be stored correctly. Additionally, give some consideration to the type of climate in which your canine toys chuckit massive shall be stored.
Another example is an Afghan hound, a good instance of a sighthound dog. Sighthound dogs rely on their sight and speed, versus scent hounds, thus the identify. It is because of this why sighthound dogs are excellent hunters. Canine who belong to this sort normally like Frisbees or balls.


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While landowners should take a moment to celebrate the cancellation of the Atlantic Coast Pipeline, (ACP), we also need to remember that now is the time to get damage repaired and the company owns easements across our land. Dominion has said that it will not ask that we return any money we have received, but it has not decided what it will repair or what to do about the easements. It could use them or sell them to others. The risk to us is that it could be claimed at some future point. We can't forget this claim on our property that supersedes our use of our property as long as it exists. Since eminent domain was threatened or used to obtain these easements for a project Federal Energy Regulatory Commission (FERC) declared to be of public benefit, it seems only fair that easements be released since they will not be used for that specific use.
Many landowners have damage from tree cutting along the easement. In many cases, the trees were cut two years ago and nothing further has been done. Normal weeds and invasive species brought in by the tree cutters are growing. Fences, gates, and driveways may have damage. There is no single solution for repairing the damage.
We are in unknown territory right now. ACP has already announced its financial loss on the pipeline project so it cannot include the value of easements in that amount. Some of the eminent domain attorneys are already working with North Carolinians to address this issue. The question has been raised of whether FERC has the authority to tell ACP to give back easements awarded it through the courts via eminent domain.
Buckingham county has already rescinded the zoning variance, the special use permit, it made to allow the compressor station. That land is once again zoned for agricultural use. The speed with which the county moved to protect the land gives us a signal about the importance of not having easements hanging over us. Like the county, we don't want to be informed years down the road that the company will act on that easement and force another project across our property without any consideration of our use of our property.
Some of us, including the Southern Environmental Law Center (SELC,) told FERC that we want our easements back when we had a 15-day opportunity to respond to ACP's request to FERC have a year to close out the project and additional time to decide what to do with the section in West Virginia.
Some people think that unless they repay the money they cannot ask for return of their easement. However, those who received money have already paid taxes on it. If they worked with an attorney, they paid attorney fees. If they went to court, they had costs for multiple property assessments, transcripts for depositions, preparation of exhibits, and travel and job absence costs, for example. Many landowners spent hours learning about what was happening and attempting to protect land. We did not ask for this pipeline project, but had many costs over the last six years without even mentioning the emotional stress, anguish and personal conflicts experienced. Many landowners did not receive enough money to come even close to paying these costs.
Right now landowners may want to take any available opportunity to express the desire to have damage repaired and to regain easement ownership to political leaders and company representatives. We need to work together and support each other through this process. Some of us may also want to work toward getting state and federal laws changed to better protect landowners in the future. Stay tuned as we work with our allies to forge a reasonable plan.
It's not over yet!
Irene Ellis Leech, Ph.D., is a Buckingham native who graduated from Buckingham County High School and Virginia Tech. She is an owner/operator of Mt. Rush Farm and is employed by Virginia Tech currently teaching Consumer Studies. She has also been an Extension Consumer Education Specialist and on the State 4-H Staff and she taught at Appalachian State University. An active consumer advocate in Virginia and nationally, she is involved in a number of organizations and coalitions. The Atlantic Coast Pipeline would have bisected the family farm for 1.1 mile and her home in Montgomery County, VA is within the evacuation zone of the Mountain Valley Pipeline.


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Sunderland legend Gordon Armstrong joined the Roker Rapport Podcast this week, and spoke candidly about his past involvements with groups who were looking to buy the club from Ellis Short.
Armstrong - who made 416 appearances for the Lads over eleven years - is now a football agent and represents a large number of players currently operating within the professional game.
He also has a role on the committee of the 'Red and White Army' supporter's group and still attends games to this day as a fan. Furthermore, due to his standing in the game both as a businessman and as a former player, he has been contacted on a variety of occasions to get involved in talks regarding potential takeovers of the football club.
During the Podcast recording one of the Roker Rapport listeners submitted a question asking Gordon if he was involved with any of the consortiums looking to buy the club, to which he responded:
No, that's not true, no. I have been involved with one or two of the consortiums (in the past) that has been involved in trying to buy it, it's never came off.
I think there is somebody locally trying to buy the club right now, so we'll see what happens, see how it moves forward.
That's the big thing that does need to change is the owner of the football club - probably for his own good as well as much as the football club's, for everybody's sanity I think it does need to change and hopefully that happens.
But yeah, I've been involved before, i.e. with the German consortium and other bits and bobs.
When prompted for further details on that particular deal, Gordon added:
They had a meeting with Mr Short and they couldn't find an agreement, basically, so that's where that ended up.
There's been loads of others I guess that have looked at it - there was another group that I was dealing with, they were very interested at one stage, but when we were in the Premier League and they dropped out - an American group.
There is very little known about the consortium in question, though in an interview back in February with Roker Report, BBC Newcastle's Nick Barnes hinted that he wouldn't rule out that the Germans could come back in for the club now once our relegation to League One has been officially confirmed.
Journalist Alan Nixon had suggested during the course of the talks that funding was a sticking point between Short and the party discussing a takeover. Ultimately the American financier issued a statement saying the bid was not in the 'best interests' of Sunderland AFC and it has been suggested he was unconvinced by the German consortium's plans for the club.
The full Podcast with Gordon Armstrong, which is available for free on iTunes, Acast and Youtube, can be found here.


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What is a pre-wedding shoot?
A pre-wedding shoot is arranged with the photographer and the couple to be married. It is a relatively inexpensive way to have a trial run with your photographer before booking them to cover your wedding day. A pre-wedding shoot is designed to create beautiful memories of your engagement, whilst also preparing you to take great, natural looking pictures on the big day.
Feeling confident in front of the camera
The main purpose of a pre-wedding shoot is to build the confidence of the couple in front of the lens and to create a good rapport with the photographer. The session should be fun, relaxing and the results should get rid of any anxiety about looking your best on the BIG day! I coach couples through a range of simple poses and techniques that make photos look natural, even when they are staged. From time to time the couple will even forget that I'm there!
Picturesque surroundings
Couples are advised to pick a picturesque location that is significant to them, or if preferred, the photographer can suggest locations that provide the perfect backdrop to their photos. I like to shoot in central London, including iconography that represents London, red phone boxes, Black taxi's Edwardian building etc. Clients are advised to dress for an evening out ("Out, out!") and often use the opportunity to go out for the evening after their photos.
Memories to cherish
Following the shoot, clients receive their best photos in high resolution. They are left with a beautiful keepsake to look back on in the years to come, that can be printed and used for save the date cards, framed prints, guest book covers, social media and much more.


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Screenshots Of Windows 10 Build 10114 Leaked Online
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Screenshots of Windows 10 Build 10114 got leaked online today. It reveals no.of new changes from the last publicly released build 10074.
1) As you can see in the image below, you now have File Explorer and Settings options above Power button in the Start Menu. Also, there is no handle to make Start Menu full screen.
2) You can customize Start Screen behavior under Settings. You can turn on/off the app suggestions that show up in the Start Menu. Also, you can select the Full-screen option here.
3) When the Start Menu is full screen, you don't have left pane which will have recently used apps list, power button and more. Instead, there is a new hamburger menu on the top left.
4) This hamburger menu of the top left will have the left pane options of Start Menu.
5) This build also includes completely revamped Windows Insider app. It has got Windows App platform treatment with hamburger menu options and more.
Source: Neowin


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Implementation Of Collaborative E-Supply-Chain Initiatives Case Study Help Analysis With Solution Online
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We discuss the challenges of implementing an Internet-based platform for creating collaborative supply chains using a case study in the retail sector. The case presents how an Internet-based collaboration platform was implemented to address the strategic issue of increasing shelf availability and customer service in grocery retailing, an issue that has emerged as one of the major confrontations for the whole sector over the past years. The case presented shows the challenges of executing such strategic collaborative supply-chain initiatives which, although arguably beneficial, can be hindered by IT adoption failures. A longitudinal view of the case is presented, from an initial pilot back in 2001 to the final success in 2005. We discuss the particular challenges of the execution of Internet-enabled collaborative supply-chain initiatives as well as possible managerial actions through a simple framework we develop based on the lessons from the case.
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Blog posts are stressful, man. So are taxes, emails, and Facebook. That's why, for the past few weeks, these other stressful tasks have taken precedence over the #1 task of any English major—reading.
Reading has not been a priority of mine for about a month now. Too many deadlines, too many applications for this job or that scholarship, too many extracurricular obligations…too many ridiculous excuses. It was only after chatting with UConn's Spring Aetna Writer-In-Residence and visiting poet G.C. Waldrep that I realized how rare reading poetry just to read it—in a context 100% unrelated to a school assignment—has been in the past few weeks.
A poet that does not read poetry. Worse yet—a poetry editor that does not read poetry.
A fraud?
No…just a confused, seemingly "stressed" (though really, my stress is all just an illusion) Type A college nerd. Eh, nothing new.
Solution? For myself and other fellow sufferers? Stop prioritizing the "little things"—yes, even our beloved Form 1040A's – EZ's—over the sacred act of reading an actual text. Reading is like a good relationship—if you like a book, then put a ring on it. Commit—immerse yourself—dedicate time to it. Marry a book—for a while or for life.
You know, blog posts are stressful, man. So I'll abruptly finish this one and encourage you and me both to—
Or, you know, watch this ONE irresistible, completely reading-related video and THEN go read a book.


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COMPUTER CONTROLLED ACOUSTIC INSTRUMENTS, PT 2 by Aphex Twin
Pros: Consistently interesting in terms of its composition
Cons: Very unconventional concepts of melody
It's a strange time to be a fan of British electronic musician, composer, and producer Richard D. James, who most often works under the pseudonym of Aphex Twin. For more than a decade, the once incredibly prolific James released virtually nothing, but since the long-delayed release of the self-titled Caustic Window album in 2014, the floodgates have apparently been opened. A new full length album called (my choice for album of the year) dropped in late 2014, and James has continued to release a veritable ton of previously-unheard music on . To be honest, I'm not even sure where to begin with regard to this almost intimidating deluge of material (some 150 tracks and counting), though it's a good problem to have after missing James' unique voice throughout the 2000s.
Though there was quite a bit of publicity surrounding Syro's release, the January 23, 2015 release of a (basically, self-explanatory) extended play called Computer Controlled Acoustic Instruments Pt 2 (there never was a part one) was a bit of a surprise – after all, two new Aphex Twin releases within a few months time would have been unheard of only a year ago. While Syro was an expansive, essentially fully-developed album however, CCAIpt2 is an almost fragmentary work that harkens back to a time in the early-to-mid '90s when James was in the midst of releasing a seemingly endless amount of imaginative and playful, occasionally outstanding material. As I'd expect from most everything bearing the Aphex Twin name, CCAIpt2 is nothing if not a consistently interesting and creative, well-produced album even if it doesn't rank among the artist's flat-out best.
It's good to know that while the electronic music scene circa 2015 is vastly different from the one in the '90s, Aphex Twin has returned to make lousy producers look bad.
Like many of James's other albums, the tracks on CCAIpt2 feature odd and sometimes unpronounceable names – which only adds to the overall strangeness of this album. "diskhat ALL prepared1mixed 13" is the discordant opening track, propelled by a tick-tocking beat and a groaning bass. Gurgling and flatulent, this first track already firmly establishes the jerky and almost sinister sound texture that exists throughout the album – particularly on the more substantial pieces. After a brief snare drum outburst, we get an alternate, even more jangly and burpy rendition of the opening number before a shorter, piano-based piece of arpeggios being slammed together. Robotic tinkling and moaning keyboard elements make "DISKREPT 4" a somewhat demented-sounding piece, while the next three tracks exist as slightly disjointed percussion experiments.
One can only imagine the amount of tweaking that went into this, or any, Aphex Twin recording.
Many of the tracks here seem to imagine what the malfunction of an elaborate grandfather clock might sound like, and several contain bizarre and sometimes creepy background ambiance – the sounds of children babbling or dogs barking for instance. I found myself looking over my shoulder a few times while listening to this album, and had to really study the mix to determine whether or not I was hearing ghosts.
Who the hell knows what the title of "disk prep calrec2 barn dance [slo]" means (many James tracks have names which refer to software or equipment used to make them), but it's another track of throbbing bass, clanging rhythmic elements and sparse, unconventional melody. Following a further deconstruction of melody (on "DISKREPT1") and rhythm (on "diskhat2"), we get the almost obligatory piece that reminds a listener of James's considerable talents as a composer. "piano un10 it happened" is positively gorgeous, a simple yet intricate piano piece that's the complete antithesis of every other track here. This composition is well worth checking out even if the others here aren't to a particular listener's taste – and let's face it, not many people would really "like" this album if it comes down to it. Closing track "hat5c0001 rec-4" shuffles along to another grumbly bass line and clanking rhythm, sounding like what Trent Reznor of Nine Inch Nails might come up with if he tried his hand at making explicitly experimental music.
In the end, Computer Controlled Acoustic Instruments Pt 2 is the sort of blatantly experimental album that will be embraced by the mainstream music press in spite of its overt weirdness because the person who made it is reputable – and almost certainly a musical genius (I find it endlessly amusing that several of the same outlets that are championing this EP trashed James's similarly abstract 2001 album drukQs). As I've pointed out many times before, mediocre James is significantly better than most people's A-game, and I'd certainly rather listen to something unique like CCAIpt2 than a generic and tiresome pop record. Still, I can't shake the notion that both this EP and Syro feel like minor works when compared to things like 1995's I Care Because You Do or the following year's Richard D. James Album… to say nothing of the sublime "Come to Daddy" and "Windowlicker" singles or the two nearly flawless Selected Ambient Works albums. Maybe I'm hoping for the impossible, but I believe that James is capable of better than what he's shown in these two latest albums. While waiting for that next bona fide masterwork however, it's nice to have things like this EP to help tide one over. It's not essential, but it is worthwhile – particularly to fans of the artist.


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The replication licensing factor (RLF) is an essential initiation factor that is involved in preventing re-replication of chromosomal DNA in a single cell cycle. In Xenopus egg extracts, it can be separated into two components: RLF-M, a complex of MCM/P1 polypeptides, and RLF-B, which is currently unpurified. In this paper we investigate variations in RLF activity throughout the cell cycle. Total RLF activity is low in metaphase, due to a lack of RLF-B activity and the presence of an RLF inhibitor. RLF-B is rapidly activated on exit from metaphase, and then declines during interphase. The RLF inhibitor present in metaphase extracts is dependent on the activity of cyclin-dependent kinases (Cdks). Affinity depletion of Cdks from metaphase extracts removed the RLF inhibitor, while Cdc2/cyclin B directly inhibited RLF activity. In metaphase extracts treated with the protein kinase inhibitor 6-dimethylaminopurine (6-DMAP), both cyclin B and the RLF inhibitor were stabilized although the extracts morphologically entered interphase. These results are consistent with studies in other organisms that invoke a key role for Cdks in preventing re-replication of DNA in a single cell cycle.
During each S phase of the eukaryotic cell division cycle, the entire genome is precisely duplicated. To achieve this, many hundreds or thousands of replication origins must each fire once and only once in each S phase. Studies using cell-free systems derived from Xenopus eggs have revealed that re-replication of chromosomal DNA in a single cell cycle is prevented by the action of two distinct replication signals (Blow and Laskey, 1988; Chong et al., 1996). The first signal, replication licensing factor (RLF)1, "licenses" chromosomal DNA by putting replication origins into an initiation-competent state. The second signal, S-phase promoting factor (SPF), induces licensed origins to initiate and, in doing so, removes the licence. To achieve precise duplication of chromosomal DNA, the licensing and initiation signals must act on the DNA sequentially, and never at the same time. This is achieved in two different ways. First, active RLF cannot cross the nuclear envelope, so it can only license DNA when the nuclear envelope has broken down in mitosis (Blow and Laskey, 1988; Leno et al., 1992; Coverley et al., 1993; Blow, 1993); in contrast, SPF can only initiate DNA replication on licensed DNA within an intact nucleus (Blow and Watson, 1987; Newport, 1987; Sheehan et al., 1988; Blow and Sleeman, 1990). Secondly, both activities are periodic in the cell cycle: RLF is abruptly activated after the metaphase–anaphase transition and decays during interphase (Blow, 1993), while SPF activity can only be detected during interphase (Blow and Nurse, 1990). The spatial separation of RLF and SPF activities is thereby enhanced by a temporal regulation.
The activation of RLF that occurs at the metaphase– anaphase transition in Xenopus can be blocked by certain protein kinase inhibitors, such as 6-dimethylaminopurine (6-DMAP), that are known to inhibit cyclin-dependent kinases (Blow, 1993; Kubota and Takisawa, 1993; Vesely et al., 1994). The licensing system has been subjected to biochemical and immunological analysis using extracts treated with these kinase inhibitors (Chong et al., 1995; Kubota et al., 1995). RLF activity can be separated chromatographically into two essential components, RLF-M and RLF-B, both of which are required for licensing (Chong et al., 1995). RLF-M has been purified to apparent homogeneity, and it consists of a complex of all six currently identified members of the Xenopus MCM/P1 family, XMcm2– XMcm7 (Chong et al., 1995, 1996; Thömmes, P., Y. Kubota, H. Takisawa, and J.J. Blow, manuscript submitted for publication). Anti-XMcm3 antibodies coprecipitated all six Xenopus MCM/P1 proteins in a complex closely resembling the RLF-M complex (Kubota et al., 1995; Madine et al., 1995a,b; Thömmes, P., Y. Kubota, H. Takisawa, and J.J. Blow, manuscript submitted for publication) and depleted the extracts of RLF-M activity (Chong et al., 1995).
MCM/P1 genes were originally identified in Saccharomyces cerevisiae in a screen for mutants unable to efficiently initiate replication at particular replication origins (Maine et al., 1984), due to a failure of the initiation process (Maiti and Sinha, 1992; Yan et al., 1993). The proteins show cell cycle–dependent changes in subnuclear localization, being observed within the nucleus only during late mitosis and G1 (Hennessy et al., 1990; Yan et al., 1993; Dalton and Whitbread, 1995). Homologous MCM/P1 genes have been identified in a wide range of eukaryotes including insects, plants, amphibians, and mammals, where they fall into six related groups designated MCM2–MCM7 (Chong et al., 1996; Kearsey et al., 1996). Consistent with their role in yeast, MCM/P1 proteins in higher eukaryotes are required for DNA replication (Kimura et al., 1994; Todorov et al., 1994; Chong et al., 1995; Kubota et al., 1995; Madine et al., 1995a; Treisman et al., 1995) and behave in accordance with their role as a central component of the licensing system. They associate tightly with chromatin during late mitosis and G1, but are removed during replication (Kimura et al., 1994; Chong et al., 1995; Kubota et al., 1995; Madine et al., 1995a,b; Todorov et al., 1995; Coué et al., 1996). Reassociation of XMcm3 with chromatin, which is required before an additional round of replication can take place, only occurs after permeabilization or breakdown of the nuclear envelope (Chong et al., 1995; Kubota et al., 1995; Madine et al., 1995a,b). The licensing of chromatin by RLF-M and RLF-B in Xenopus requires the presence of the Xenopus origin recognition complex (ORC) on the chromatin (Rowles et al., 1996).
In this paper we analyze the cell cycle control of RLF activity in Xenopus egg extracts. Total RLF activity is sharply periodic in the cell cycle. An RLF inhibitor is present in metaphase extracts and appears to be directly dependent on the activity of cyclin-dependent kinases. When the inhibitor is removed or diluted away, RLF-M, but not RLF-B, activity can be detected. RLF-B, but not RLF-M, activity is unstable in interphase and decays over a period of 60–120 min. However, the decay of RLF-B activity in interphase is not associated with activation of an RLF inhibitor and does not depend on cyclin-dependent kinases.
Materials And Methods
Preparation and Use of Xenopus Egg Extract
Metaphase-arrested and interphase Xenopus extract was prepared as described (Blow, 1993) and stored in 20-μl aliquots in liquid nitrogen. Just before use, extract was thawed at room temperature and supplemented with 250 μg/ml cycloheximide, 25 mM phosphocreatine, and 15 μg/ml creatine phosphokinase. Metaphase-arrested extract was released into interphase by the addition of 0.3 mM CaCl2. To block RLF activation, metaphase-arrested extracts were supplemented with 3 mM 6-DMAP and mixed thoroughly before CaCl2 addition (6-DMAP–treated extract). For replication assays, extract was supplemented with 1/200 vol 10 mCi/ml [α32P]dATP (Amersham Intl., Little Chalfont, UK). Extract incubations were performed at 23°C.
Suc1 depletion was performed essentially as described (Blow and Nurse, 1990). Metaphase extract was mixed with an equal volume of p13suc1-coupled Sepharose (10 μg Suc1/ml beads, washed in LFB1 [40 mM Hepes KOH, pH 8.0, 20 mM K2HPO4/KH2PO4, pH 8.0, 2 mM MgCl2; 1 mM EGTA, 2 mM DTT, 10% sucrose, 1 μg/ml each of leupeptin, pepstatin, and aprotinin] and incubated on a rotating wheel at 4°C for 60 min. The supernatant was recovered by spinning the bead/extract slurry past a glass ball wedged in a sawn-off yellow Gilson tip in an Eppendorf tube.
Preparation of DNA Templates
Demembranated Xenopus sperm nuclei were prepared by lysolecithin treatment as described (Blow and Laskey, 1986). They were assembled into chromatin in 6–DMAP-treated extract, to form "6-DMAP chromatin" as follows (Chong et al., 1995, 1997): 80 μl of a metaphase-arrested extract was supplemented with phosphocreatine, creatine phosphokinase, 3 mM 6-DMAP, and CaCl2 as described above, and then supplemented with 1.3 × 106 demembranated sperm nuclei (∼50 ng DNA per μl extract). The mixture was incubated for 12 min at 23°C, and then diluted in 1 ml NIB (50 mM KCl; 50 mM Hepes KOH, pH 7.6; 5 mM MgCl2; 2 mM DTT, 0.5 mM spermidine 3HCl; 0.15 mM spermine 4HCl; 1 μg/ml each leupeptin, aprotinin, and pepstatin) and underlayered with 100 μl NIB supplemented with 15% sucrose. The chromatin was isolated by centrifugation in a swing-out rotor at 6,200 g for 5 min at 4°C. The pellet was resuspended in 45 μl NIB (to a final concentration of 80 ng DNA/per μl) and frozen in 5-μl aliquots in liquid nitrogen.
Preparation of RLF-B and RLF-M
Polyethylene glycol (PEG)–fractionated RLF-B and RLF-M were prepared as described (Chong et al., 1995, 1997). Eggs were dejellied in 2% cysteine solution, washed three times in Barth solution (88 mM NaCl, 2 mM KCl, 1 mM MgCl2, 15 mM Tris HCl, pH 7.4, 0.5 mM CaCl2), and then incubated for 5–10 min in 100 ml Barth plus 20 μl 10 μg/ml calcium ionophore A23187. The activated eggs were then washed three times in Barth solution at 23°C and three times in EB (50 mM KCl, 50 mM Hepes KOH, pH 7.6, 5 mM MgCl2, 2 mM DTT) at 4°C. All additional steps were performed at 4°C or on ice. Eggs were packed by centrifuging at 800 g in a swing-out rotor for 1 min. Excess buffer and sick eggs, which float to the surface during this treatment, were then removed. Packed eggs were spincrushed by centrifugation at 10,000 g for 10 min in a swing-out rotor. The cytoplasmic layer was taken, supplemented to a final concentration of 10 μg/ml cytochalasin B and 15% EDB-S (50 mM KCl, 50 mM Hepes KOH, pH 7.6, 10% sucrose, 2 mM DTT, 0.4 mM MgCl2, 0.4 mM EGTA, 1 μg/ml each of pepstatin, leupeptin, and aprotinin), and then diluted with 4 vol of LFB1 containing 50 mM KCl and 0.5 mM freshly added PMSF. This diluted extract was spun at 60,000 g for 20 min at 4°C in a swing-out rotor. The supernatant ("licensing factor extract") was decanted, drop frozen in liquid nitrogen, and stored at −70°C. Since it is fivefold diluted over neat extract, licensing factor extract is designated a concentration of 0.2×.
Differential PEG precipitation was performed as follows (Chong et al., 1995, 1997): licensing factor extract was supplemented with 0.075 vol of a 50% PEG solution (50% PEG 6000 [BDH Chemicals Ltd., Poole, UK] in LFB1) to give a final concentration of 3.5% PEG. Extract was incubated on ice for 30 min, and then centrifuged at 10,000 g for 10 min in a fixed- angle rotor. The supernatant was decanted, and residual PEG solution was removed after an additional brief spin. The pellet was then resuspended in LFB1 at 1/25 of the original volume of licensing factor extract to give 5× concentrated RLF-B, and was stored in aliquots at −70°C. The supernatant was supplemented with an additional 0.11 vol of a 50% PEG solution, giving a final concentration of 9% PEG, incubated on ice, and pelleted as before. The pellet was resuspended in LFB1 at 1/25 of the original volume, to give 5× concentrated RLF-M, and was stored in aliquots at −70°C.
RLF-M was further fractionated on Q Sepharose to produce RLFMinter as described (Chong et al., 1995, 1997). RLF-M produced by PEG precipitation was diluted to 1× in LFB1 plus 100 mM KCl and was adsorbed in batch for 30 min at 4°C onto an equal volume of loose Q Sepharose (Pharmacia, Uppsala, Sweden) preequilibrated in LFB1 plus 100 mM KCl. The media was then packed into a column, washed with LFB1 plus 100 mM KCl, and eluted as a step in LFB1 plus 325 mM KCl. Eluted material was reprecipitated with PEG and resuspended in LFB1. RLF-Mmeta was produced in the same way as RLF-Minter, except that the starting material was metaphase-arrested extract.
For the maturation promoting factor (MPF) inhibition experiment (see Fig. 4 b), RLF-M from the Q Sepharose step was further purified by phenyl Sepharose chromatography and gel filtration, as previously described (Chong et al., 1995, 1997). For this experiment, the RLF-B preparation was also further purified, according to the protocol previously described for Xenopus ORC (Rowles et al., 1996; Chong et al., 1997). Briefly, PEGfractionated RLF-B was bound to phosphocellulose equilibrated in LFB1 plus 150 mM KCl, and then step-eluted in LFB1 plus 500 mM KCl. Eluted material, containing both RLF-B and Xenopus ORC activity, was precipitated with 40% ammonium sulfate, and the pellet was resuspended in LFB1 for assay.
Licensing Assay
RLF was assayed in a two-step protocol as described (Chong et al., 1995, 1997). 2-μl samples to be assayed were incubated with 0.3 μl 6-DMAP chromatin (80 ng DNA/μl) for 15 min for the "licensing reaction." In assays for individual components, the 2-μl samples were typically composed of 1-μl fraction of interest, plus 1-μl PEG-cut RLF-B and/or RLF-M diluted to 0.5× in LFB1, plus 50 mM KCl and 2.5 mM Mg-ATP. For extract dilution experiments, dilutions were performed with LFB1 plus 50 mM KCl. Once the licensing reaction was finished, 5.7 μl 6-DMAP–treated extract containing cycloheximide, phosphocreatine, creatine phosphokinase, 6-DMAP, CaCl2 and [α32P]dATP were added. The incubation was continued for an additional 90 min at 23°C (the "replication reaction"). Replication reactions were terminated by the addition of 150 μl Stop-C (20 mM Tris HCl, pH 7.5, 5 mM EDTA, 0.5% SDS) plus 0.2 μg/ml proteinase K and incubated at 37°C for 15 min. TCA precipitation was performed as described (Blow and Laskey, 1986; Chong et al., 1997), and the total DNA synthesized (in ng DNA/μl) was calculated, assuming 50 μM endogenous dATP in the extract (Blow and Laskey, 1986). Bromodeoxyuridine triphosphate (BrdUTP) density substitution was performed as described (Blow and Laskey, 1986).
Alkaline Agarose Gel Electrophoresis
Reactions for agarose gel electrophoresis were stopped by digesting with Stop-N (20 mM Tris-Cl, 200 mM NaCl, 5 mM EDTA, 0.5% SDS, pH 8.0) containing 2 μg/ml RNase A and 200 μg/ml Proteinase K for 30 min at 37°C. DNA was extracted sequentially with phenol, phenol-chloroform, and chloroform, and then precipitated with ethanol. Pellets were resuspended in alkali gel loading buffer (50 mM NaOH, 1 mM EDTA, 1.25% Ficoll, 0.0125% bromocresol green). Alkaline gels were prepared by adding the required amount of agarose to 50 mM NaCl and 1 mM EDTA. Once set, the gels were allowed to equilibrate for 1 h in alkaline gel running buffer (30 mM NaOH, 1 mM EDTA), and then were run at 2 V/cm. After electrophoresis was complete, gels were fixed in 7% TCA, dried, and autoradiographed.
Cip1 and Cdc2/Cyclin B
Glutathione-S-transferase (GST)–tagged Cip1 was prepared as described (Strausfeld et al., 1994). It was added to Xenopus extracts at 150 nM, an amount sufficient to inhibit DNA replication by >95% (Strausfeld et al., 1994). Purified Cdc2/cyclin B was purchased from Upstate Biotechnology Inc.(Lake Placid, NY) and was used in the licensing reaction at a final concentration of 1.25 ng/μl.
Antibodies and Western Blotting
For Western blotting, chromatin was isolated from extract as described for the preparation of 6-DMAP chromatin, except that the NIB buffer was supplemented with 0.1% NP-40 (Chong et al., 1995, 1997). Chromatin pellets were resuspended in gel loading buffer, electrophoresed, and Western blotted by standard techniques. Anti-XMcm3 rabbit polyclonal antibodies were raised against recombinant GST-tagged XMcm3 (amino acids 423– 798) produced from the construct described by Madine et al. (1995a).
35S Labeling and Autoradiography
200 μCi [35S]methionine (Amersham Intl.) was dried under vacuum and redissolved in 40 μl metaphase-arrested extract supplemented with phosphocreatine and creatine phosphokinase. The extract was incubated for 30 min at 23°C to label newly synthesized proteins, before the addition of 250 μg/ml cycloheximide. Extract was then supplemented with various concentrations of 6-DMAP and then with 0.3 mM CaCl2, before being incubated for an additional 30 min at 23°C to allow cyclin degradation to occur. Cyclin–cyclin-dependent kinase (Cdk) complexes were then collected on p13suc1 beads at 4°C for 45 min. The beads were washed several times, and then were resuspended in sample loading buffer for electrophoresis and autoradiography according to standard techniques.
Quantification of RLF-M Required for DNA Replication
RLF-M consists of a complex of MCM/P1 proteins, including XMcm3 (Chong et al., 1995). To correlate the amount of RLF-M activity in Xenopus extracts with the amount of XMcm3 protein, we performed quantitative Western blotting on extracts and on chromatin assembled in these extracts (Fig. 1,a). An estimate of 75–150 μg XMcm3 protein per ml Xenopus extract (0.75–1.5 μM) was provided by a number of duplicate blots (e.g., Fig. 1,a, lanes 1 and 2 and 17–19). Fig. 1,a also quantifies the association of XMcm3 with chromatin. Demembranated Xenopus sperm nuclei (Fig. 1, lanes 10–16) or chromatin previously assembled in 6-DMAP–treated extract (6-DMAP chromatin; Fig. 1, lanes 3–9) were briefly incubated with serial dilutions of Xenopus egg extract. Chromatin was then isolated by centrifugation through sucrose and immunoblotted for XMcm3. As more extract was incubated with a fixed quantity of chromatin, the amount of chromatin-associated XMcm3 increased. Only at the highest concentrations of DNA (6–12 ng DNA per μl extract) was there any sign of the chromatin becoming saturated with XMcm3. Comparison with the recombinant XMcm3 standard (Fig. 1, lanes 1 and 2) suggests that the extract can assemble in excess of 30 ng XMcm3 onto 300 ng DNA, equivalent to one XMcm3 molecule bound to every 1.5 kb of DNA on average. Previous studies have estimated that the replicon size of the early Xenopus embryo is 10–20 kb (Blow and Watson, 1987; Mahbubani et al., 1992; Hyrien and Méchali, 1993), suggesting that there may be >10 XMcm3 molecules bound to each replicon.
Fig. 1,b examines the RLF activity of chromatin licensed with different dilutions of interphase extract. Chromatin was licensed with serial dilutions of extract, and then transferred to 6-DMAP–treated extract (lacking RLF activity) supplemented with [α32P]dATP. At different times after transfer, the amount of DNA synthesis was measured. When incubated in extract at concentrations of above 1/4–1/8 (corresponding to 100–200 ng DNA per μl neat extract), chromatin was efficiently licensed, leading to almost complete replication after an additional 90-min incubation in 6-DMAP–treated extract. This is similar to replication rates seen in untreated extract. When licensed with lower amounts of extract (1/16 dilution and below), the chromatin replicated at a lower rate, as though only a fraction of the replication origins had initiated. Comparison of Fig. 1 a and 1 b suggests that maximal replication rates were still seen with subsaturating quantities of XMcm3 assembled onto the chromatin.
Cell Cycle Variation in RLF Activity
The Xenopus egg is arrested at metaphase of meiosis II. At fertilization, a calcium wave is released that overcomes the metaphase arrest, and the early embryo embarks on a series of 12 rapid cell cycles. Extracts prepared from unfertilized eggs maintain the metaphase arrest and can be released into interphase by addition of CaCl2 (Lohka and Masui, 1984). RLF activity is low in such metaphase- arrested extracts, but it rapidly increases upon CaCl2 addition (Blow, 1993). We first determined the quantity of RLF-B and RLF-M activity present in metaphase-arrested and in interphase egg extracts (Fig. 2). Chromatin was licensed with serial dilutions of extract and transferred to 6-DMAP–treated extract, and the total DNA synthesized over a 90-min incubation was measured (Fig. 2, filled circles). Consistent with the time courses shown in Fig. 1,b, interphase extract efficiently licensed DNA synthesis after dilution as low as 1/4–1/8 (Fig. 2,b). In contrast, no RLF activity was provided by the metaphase-arrested extract (Fig. 2 a).
The titrations of RLF activity in metaphase and interphase extracts were repeated in the presence of exogenous RLF-B (Fig. 2, open circles) or RLF-M (Fig. 2, open squares) to respectively determine the RLF-M and RLF-B activity of the samples. Titrations of activity in interphase extract under these conditions remained virtually unchanged (Fig. 2,b), suggesting that these extracts had approximately equal activities of RLF-M and RLF-B. At high concentrations of metaphase extract, addition of exogenous RLF-B or RLF-M induced no RLF activity (Fig. 2,a). However, when metaphase extracts were diluted down to 1/8–1/16, significant RLF-M activity could be detected. No RLF-B activity was observed at any dilution of metaphase extract. The appearance of RLF-M activity after dilution of metaphase extract was unexpected and suggested the presence of an RLF inhibitor in these extracts that was being diluted away until it no longer inhibited the reaction. The presence of an RLF inhibitor was demonstrated by mixing serial dilutions of metaphase extract with a constant amount of active RLF (RLF-B plus RLF-M; Fig. 2,a, triangles). The licensing activity of exogenous RLF was inhibited by high concentrations of metaphase extract. When metaphase extracts were diluted further, the inhibitory activity was titrated away, allowing exogenous RLF to license chromatin. The disappearance of the inhibitory activity upon dilution corresponded to concentrations where RLF-M activity became assayable. In contrast, no RLF inhibitor was observed in interphase extracts (Fig. 2 b, triangles). The simplest explanation for the ability to assay RLF-M activity after dilution of metaphase extract is that the RLF inhibitor affects only RLF-B activity. Consistent with this interpretation, we could find no evidence for the inhibition of RLF-M activity in metaphase (see below).
To quantify cell cycle variations of RLF activity, extract was incubated for various times, and then diluted to 1/8, where RLF activity should be in the linear range for the assay (Fig. 3). In these extracts, chromatin decondensation and nuclear assembly are complete by ∼30–40 min after CaCl2 addition, while S phase occurs at 40–90 min (Blow, 1993). RLF levels in metaphase were low, but, within 15 min after release from the metaphase arrest by CaCl2 addition, maximum levels of total RLF activity were observed (Fig. 3,a, filled circles). Total RLF activity decayed over 1–2 h (Fig. 3,b, filled circles), although the kinetics of the decay varied from extract to extract. This decay of total RLF activity was almost entirely due to decay of RLF-B activity (Fig. 3 b, open squares), while RLF-M activity remained fairly constant throughout the cell cycle (open circles).
Cdk Dependence of the RLF Inhibitor
The above results suggest the presence of an RLF inhibitor in metaphase extracts. Interphase Xenopus extracts can be induced to enter metaphase by addition of MPF, a Cdk activity that can be provided by either Cdc2/cyclin B (Labbé et al., 1989a; Gautier et al., 1990) or Cdc2/cyclin A (Luca et al., 1991; Roy et al., 1991). We have previously shown that the addition of high levels of cyclin A to interphase Xenopus extracts inactivates RLF (Blow, 1993) and blocks DNA replication (Strausfeld et al., 1996). We therefore examined whether the RLF inhibitor present in metaphase extracts was dependent on Cdk activity. Cyclin-dependent kinases can be affinity depleted from Xenopus extracts using p13suc1 coupled to Sepharose beads (Dunphy et al., 1988). When p13suc1 depletion is performed on metaphase-arrested Xenopus extracts, subsequent DNA synthesis is blocked (Blow and Nurse, 1990). Fig. 4,a (filled circles) shows that p13suc1 depleted metaphase extracts lack RLF activity. However, unlike the undepleted extracts (Fig. 2,a), RLF-M activity could be detected at the highest extract concentrations (Fig. 4,a, open circles). Thus, no RLF inhibitor could be detected in these extracts (Fig. 4,a, triangles), and the lack of RLF activity was apparently due only to a lack of RLF-B activity (Fig. 4 a, squares).
The Xenopus early embryo has at least three distinct cyclin-dependent kinases: Cdc2/cyclin A, Cdc2/cyclin B, and Cdk2/cyclin E. Similar to the effect of p13suc1 depletion, immunoprecipitation of Cdc2 from metaphase extracts blocked activation of RLF-B (data not shown) and prevented subsequent DNA replication (Blow and Nurse, 1990). To demonstrate a direct role for Cdc2 in inhibiting RLF, we prepared purified RLF-M (Chong et al., 1995) and partially purified RLF-B (Rowles et al., 1996; Chong et al., 1997). Incubation of recombinant Cdc2/cyclin B with these purified fractions caused strong inhibition of licensing (Fig. 4 b). This result strongly suggests that Cdc2/cyclin B can directly inhibit the RLF system. Consistent with this idea, we show below that stabilization of the RLF inhibitor correlates well with stabilization of cyclin B.
We next performed a partial (∼50-fold) purification of RLF-M from metaphase and interphase extracts to investigate whether active RLF-M can be found in a metaphase-arrested extract (Fig. 5,a). RLF-M prepared from metaphase extracts (RLF-Mmeta) showed virtually identical activity to RLF-M prepared from interphase extracts (RLF-Minter). Mixing RLF-Mmeta and RLF-Minter together showed that the RLF-M had been purified away from the metaphase-specific inhibitory activity. At this stage we cannot be completely certain that RLF-M is fully active in metaphase extracts, as it is possible that an inhibitory modification (such as phosphorylation) might be reversed during the purification protocol. In metaphase Xenopus extracts, the XMcm4 protein, a component of RLF-M (Chong et al., 1995; Thömmes, P., Y. Kubota, H. Takisawa, and J.J. Blow, manuscript submitted for publication), is phosphorylated and migrates slower on SDS gels (Fig. 5,b, lanes 1 and 3; Coué et al., 1996). This phosphorylation can be blocked with phosphatase inhibitor okadaic acid (Fig. 5,b, lane 7; Coué et al., 1996), but is lost during the purification of the RLF-Mmeta fraction (Fig. 5,b, lane 2). However, the presence of the slow migrating form of XMcm4 does not correlate with the presence of the RLF inhibitor, since XMcm4 is fast migrating in 6-DMAP– treated extract (Fig. 5, lane 6) where the RLF inhibitor is present (see below). Furthermore, XMcm4 is still slow migrating in metaphase extracts diluted to 1/8 and incubated with RLF-B (Fig. 5,b, lanes 8 and 9), under which conditions the inhibitor has been titrated away (Fig. 2,a). Conversely, most of the XMcm4 is fast migrating in Suc1- depleted extracts (Fig. 5 b, lane 5) where the inhibitor is absent. Thus, we can find no evidence that mitotic phosphorylation of RLF-M inhibits its activity.
Since the RLF inhibitor present in metaphase extracts is dependent on cyclin-dependent kinases, we next investigated whether the decline in RLF activity during interphase is also dependent on cyclin-dependent kinases. Both cyclin A and cyclin B are abruptly degraded during late mitosis, and must be resynthesized during the next cell cycle before entry into mitosis. Therefore, treatment of Xenopus egg extracts with protein synthesis inhibitors such as cycloheximide blocks the reappearance of these two cyclins. However, consistent with our previous results (Blow, 1993), cycloheximide only slightly delayed the decay of RLF that normally occurs during interphase (Fig. 6,a, halffilled diamonds). In contrast to cyclins A and B, cyclin E remains at approximately constant levels throughout the early embryonic cell cycles (Rempel et al., 1995; Chevalier et al., 1996). Treatment of Xenopus extracts with the Cdk2 inhibitor Cip1 inhibits the SPF activity of Cdk2/cyclin E and thus blocks the initiation of DNA replication (Strausfeld et al., 1994; Jackson et al., 1995). However, addition of Cip1 to cycloheximide-treated extracts did not stabilize RLF activity in the cell cycle (Fig. 6 a, filled diamonds). Similarly, affinity depletion of Cdks from interphase extract using p13suc1-coupled Sepharose did not stabilize RLF activity (data not shown). These results suggest that the decay of RLF activity during interphase is independent of Cdk activity.
Fig. 6,b shows the quantity of RLF-B and RLF-M activity in interphase extracts that had been incubated at 23°C for 2 h. Total RLF activity was reduced approximately eightfold over extract that had recently been released from metaphase (compare Figs. 2,b and 6 b, filled circles). This was almost exclusively due to a loss of RLF-B activity (open circles), while RLF-M activity remained high (squares). Despite the decline in RLF-B activity, no RLF inhibitor could be detected (Fig. 6 b, triangles). Thus, Xenopus extracts can be defective in RLF activity in at least two distinct ways. In metaphase, a potent inhibitor of RLF activity is present, and this inhibitor is directly dependent on the presence of Cdk activity. If this inhibitor is diluted away, RLF-M but not RLF-B activity can be detected. In contrast, when RLF-B activity decays during late interphase, this decay does not require Cdk activity and no RLF inhibitor becomes activated.
Role of RLF in the Initiation of Replication
6-DMAP–treated extracts lack active RLF and therefore cannot replicate double-stranded DNA (Blow, 1993). Despite this fact, they are still competent to support complementary strand synthesis on single-stranded DNA and to elongate replication forks stalled with aphidicolin (Blow, 1993). This suggests that RLF activity is required for the initiation, but not the elongation, of replication forks. However, Yan and Newport (1995) have suggested on the basis of immunofluorescence data that 6-DMAP–treated extracts are able to support the synthesis of DNA primers at replication origins, but that these primers are not correctly processed into functional replication forks. We therefore analyzed the residual DNA synthesis seen in 6-DMAP–treated extracts by alkaline gel electrophoresis (Fig. 7,a) and BrdUTP density substitution (Fig. 7, b and c). 6-DMAP treatment of the metaphase-arrested extract inhibited subsequent DNA synthesis by 95%. However, BrdU density substitution showed that all of the residual DNA synthesized in the 6-DMAP–treated extract (Fig. 7,b) was of the same density as DNA synthesized in untreated extract (Fig. 7,c), as expected of fully replicated heavy/light DNA produced by semiconservative replication. This suggests that a small number of replicons escape the 6-DMAP inhibition and produce relatively large tracts of fully replicated DNA. This conclusion was confirmed by analyzing nascent strand length by alkaline agarose gel electrophoresis (Fig. 7,a). In both 6-DMAP–treated (lane 1) and untreated extract (lane 9), virtually all of the nascent DNA was in the form of high molecular weight DNA (>23,000 nt in size). In contrast, when replication forks are stalled close to the origin by treating extracts with aphidicolin, most of the nascent DNA is observed in a broad smear of between 100 and 300 nt in length (Fig. 7,a, lanes 5–8). These results strongly suggest that 6-DMAP blocks the initiation of DNA replication at a stage before significant primer synthesis has occurred. The few replication forks that do initiate in 6-DMAP–treated extracts then proceed to synthesize extensive stretches of nascent DNA. No low molecular weight products are observed when chromatin is preincubated with RLF-B, RLF-M, or a mixture of both before incubation in 6-DMAP–treated extract (Fig. 7 a, lanes 3–5), suggesting that both of these activities are required for efficient initiation of DNA replication in 6-DMAP– treated extracts.
Effect of 6-DMAP on the Licensing System
When 6-DMAP is added to metaphase-arrested Xenopus extracts, MPF activity is inhibited, and the extracts spontaneously exit from metaphase into interphase without the need for CaCl2 addition (Zhang and Masui, 1992; Blow, 1993). These 6-DMAP–treated extracts assemble DNA into normal nuclei and, with the exception of RLF, contain all the activities required for complete chromosome replication (Blow, 1993). Similar results have been reported for two other kinase inhibitors: staurosporine (Blow, 1993; Kubota and Takisawa, 1993) and olomoucine (Vesely et al., 1994). For all these inhibitors, there is a good correlation between the inhibition of MPF activity (as judged by spontaneous assembly of interphase nuclei) and the inhibition of RLF activity. To cause inhibition of the licensing system, the kinase inhibitors must be added to Xenopus extracts before exit from metaphase arrest (Blow, 1993; Kubota and Takisawa, 1993). This suggests that the protein kinase inhibitors block the RLF activation that normally occurs upon exit from metaphase, rather than inhibiting the action of active RLF. This conclusion is confirmed in Fig. 8 a. Active RLF-M and RLF-B fractions prepared from interphase Xenopus extract by differential PEG precipitation were resuspended in buffers plus or minus 3 mM 6-DMAP. This concentration of 6-DMAP is sufficient to block RLF activation in metaphase extracts (Blow, 1993). Efficient licensing of DNA replication required both RLFB and RLF-M, but it was not significantly affected by the presence of 6-DMAP.
To measure RLF-B, RLF-M, and RLF inhibitor activity when 6-DMAP is added to metaphase-arrested extracts, we performed serial dilutions of 6-DMAP–treated metaphase extract using buffer supplemented with an additional 3 mM 6-DMAP (Fig. 8,b). The results of the titration were strikingly similar to those of untreated metaphase extracts (compare Figs. 8,b and 2 a). RLF activity was not detected at any dilution (Fig. 8 b, filled circles), but an RLF inhibitor was detected at higher concentrations (triangles). Although no RLF-B could be detected at any dilution (squares), significant RLF-M activity could be detected once the inhibitor had been diluted away (open circles). It therefore appears that although the 6-DMAP–treated extracts have morphologically exited from metaphase into interphase, they still contain the RLF inhibitor normally present in metaphase extracts.
Since the RLF inhibitor is dependent on the presence of Cdk activity, we determined whether Cdc2/cyclin B is present in 6-DMAP–treated extracts. Cyclins A and B are normally degraded on exit from mitosis. Fig. 9,a shows an autoradiograph of 35S-labeled cyclin B in extracts released from metaphase by CaCl2 in the presence of different concentrations of 6-DMAP. Consistent with previous reports (Felix et al., 1989; Luca and Ruderman, 1989), 6-DMAP at concentrations of above 2 mM blocked cyclin B degradation that normally occurs after CaCl2 addition (Fig. 9,a, lanes 5–8). However, these extracts all entered interphase as judged by the assembly of DNA into interphase nuclei, despite the stability of cyclin B at higher concentrations (Fig. 9,b; +CaCl2). Above 1 mM, 6-DMAP could inhibit MPF and cause metaphase extract to spontaneously enter interphase (as judged by the assembly of DNA into interphase nuclei) in the absence of added CaCl2 (Fig. 9,b, −CaCl2; Zhang and Masui, 1992). The concentrations of 6-DMAP just sufficient to block cyclin B degradation correlated well with concentrations at which RLF became stably inhibited (Fig. 9 c). It therefore seems most likely that while high levels of Cdc2 kinase are required to trigger cyclin B degradation (Felix et al., 1989), lower levels of kinase sufficient to inhibit RLF activity are still present in extracts treated with 3 mM 6-DMAP.
RLF is an essential initiation factor that ensures that chromosomal DNA is not re-replicated in a single cell cycle. In this paper we measure the RLF activity present in Xenopus extracts and show that the ability of extracts to license chromatin varies throughout the cell cycle. In metaphase extract, RLF-M but not RLF-B activity can be assayed, and, in addition, an RLF inhibitor is present. This RLF inhibitor is dependent on the presence of cyclin-dependent kinases and is stabilized when cyclin B is stabilized by the protein kinase inhibitor 6-DMAP. RLF activity is rapidly activated upon exit from metaphase, and then decays during late interphase. The decay of RLF activity in late interphase is mainly due to decay of RLF-B activity and is not associated with reactivation of the RLF inhibitor.
RLF Activity of Xenopus Egg Extracts
Interphase Xenopus extract can license ∼100–200 ng DNA per μl extract for efficient replication. Titration of RLF-M and RLF-B activity suggested that these two activities were present at approximately equal levels. Chromatin licensed at 100 ng DNA per μl extract could replicate at rates typical of Xenopus egg extracts. Western blotting of XMcm3, a component of RLF-M (Chong et al., 1995), suggests that, at 100 ng DNA per μl extract, one XMcm3 molecule is assembled on average onto every 5–10 kb DNA. This represents on average one to three XMcm3 molecules per replicon. However, at lower concentrations of DNA, more XMcm3 can be assembled onto chromatin (up to one XMcm3 molecule per 1.5 kb, or 10 XMcm3 molecules per replicon) without significantly affecting the overall replication rate. This higher density of XMcm3 is more likely to reflect conditions in vivo, since up to the midblastula transition (when zygotic transcription starts), the concentration of chromosomal DNA is <25 ng/μl. A similar excess of Mcm3 over replication origins has been reported in human cells (Burkhart et al., 1995) and budding yeast (Lei et al., 1996).
Cell Cycle Variation of RLF Activity
RLF activity varies throughout the in vitro cell cycle. In metaphase, RLF is largely inactive but is rapidly activated after the metaphase–anaphase transition; during interphase, activity then gradually declines (Blow, 1993). We have analyzed some of the basic causes of this variation. In metaphase an RLF inhibitor was present that inhibited licensing by exogenous RLF. When this metaphase extract was diluted so that the inhibitor no longer interfered with the licensing assay, levels of RLF-M were detected comparable with those seen in interphase extract. In contrast, no RLF-B activity was detected. Partially purified RLF-M prepared from metaphase extracts (RLF-Mmeta) was also fully active. XMcm4, a component of RLF-M (Thömmes, P., Y. Kubota, H. Takisawa, and J.J. Blow, manuscript submitted for publication), was phosphorylated during metaphase (Coué et al., 1996), but this phosphorylated XMcm4 was still associated with active RLF-M. The simplest explanation for these results is that the target of the RLF inhibitor is RLF-B, not RLF-M, although we cannot completely rule out an inhibitory modification to RLF-M that we have been unable to stabilize.
The decline of RLF activity that occurs during interphase causes a different defect than is seen in metaphase extracts. RLF-M activity remains high, but RLF-B activity declines. Unlike metaphase extracts, the loss of RLF-B activity during interphase occurs without the appearance of an RLF inhibitor. At present we cannot be certain whether the decline of RLF-B activity during interphase is of physiological relevance or whether it is an artifact of the cell-free system. However, the decline of RLF-B activity is fairly specific, since extracts retain RLF-M activity and remain competent to replicate previously licensed chromatin (Blow, 1993).
Role of RLF in the Initiation of Replication
The initiation of chromosomal DNA replication is likely to consist of several steps, including the unwinding of origin DNA, the synthesis of short DNA primers, and the establishment of a mature replication fork. On the basis of immunofluorescence data showing that 6-DMAP–treated extracts are able to support low levels of DNA synthesis, it has been suggested that RLF is not required for the synthesis of DNA primers at replication origins, but it is required for correct assembly of replication forks (Yan and Newport, 1995). We have examined this point in detail to determine whether RLF is required for the synthesis of DNA primers early in initiation. Alkaline gel electrophoresis of nascent strands showed that in 6-DMAP– treated extracts lacking RLF, no short DNA primers could be observed. In contrast, short nascent strands could be readily detected when replication forks were blocked by the polymerase inhibitor aphidicolin. Both RLF-B and RLF-M were required for efficient DNA replication, but in the presence of both these activities, nascent strands were rapidly extended into high molecular weight DNA. We therefore conclude that RLF is required for the initiation of DNA replication before the synthesis of DNA primers.
The RLF Inhibitor Depends on Cdk Activity
Cdks are key regulators of cell cycle progression (for review see Nigg, 1995). MPF is an activity present in metaphase cells capable of inducing entry into mitosis. It consists of a Cdc2/cyclin B heterodimer (Dunphy et al., 1988; Gautier et al., 1988, 1990; Labbé et al., 1989a,b). Cdc2/cyclin A, which is also active at this stage of the cell cycle, can also generate MPF activity (Luca et al., 1991; Roy et al., 1991; Strausfeld et al., 1996). The RLF inhibitor present in metaphase extracts appears to be directly dependent on Cdks active at this time. When Cdks were affinity depleted from metaphase extracts using p13suc1, the RLF inhibitor was removed. Inhibition of RLF function by kinase inhibitors such as 6-DMAP caused persistence into interphase state of the RLF inhibitor normally only present during metaphase. This also correlated with inhibition of MPF activity (Blow, 1993; Kubota and Takisawa, 1993; Vesely et al., 1994) and stabilization of cyclin B. Active RLF was also inhibited by the activation of Cdks. When recombinant cyclin A was added to interphase extract, RLF activity abruptly decayed (Blow, 1993). Furthermore, purified Cdc2/cyclin B directly inhibited licensing activity provided by purified RLF-M and partially purified RLF-B. These results strongly suggest a direct inhibition of the licensing system by Cdc2 kinase. Although MCM/P1 polypeptides are expected to be good substrates for this kinase (Coué et al., 1996), the results discussed above suggest that inhibition of licensing is unlikely to be mediated by inhibition of RLF-M function. Instead, RLF-B appears to be a more likely target of Cdk inhibition.
Inhibition of RLF function by Cdks is consistent with experiments in other organisms that show, when Cdk activity is inhibited in G2 cells, re-replication of chromosomal DNA can occur without cells passing into mitosis. This effect is plausibly caused by reactivation of RLF and relicensing of replicated DNA. Re-replication of chromosomal DNA is seen in Saccharomyces pombe cells containing a temperature-sensitive cdc2 gene when they are shifted to the nonpermissive temperature during G2 (Broek et al., 1991). Repeated rounds of DNA synthesis are seen in S. pombe cells lacking the cdc13 (cyclin B) gene (Hayles et al., 1994) or cells that overexpress the Cdc2 inhibitor Rum1 (Moreno and Nurse, 1994). In S. cerevisiae, the establishment of a pre-replicative footprint over origins of replication that probably corresponds to the licensed state can only occur in late mitosis and G1 when Cdk levels are low (Diffley et al., 1994; Piatti et al., 1996). Furthermore, inhibition of Cdks in G2/M-phase S. cerevisiae cells is sufficient to induce a pre-replicative footprint over origins of replication (Dahmann et al., 1995). Treatment of mammalian cells with certain protein kinases that inhibit Cdks was sufficient to allow re-replication of DNA in the absence of mitosis (Usui et al., 1991). Similar treatments allow mammalian G2 nuclei to become relicensed for replication in Xenopus egg extracts (Coverley et al., 1996). A requirement for Cdks to prevent re-replication of DNA late in the cell cycle therefore appears widespread throughout the eukaryotic kingdom and is plausibly mediated by an RLF inhibitor that prevents relicensing of replicated DNA. This is consistent with the results presented here demonstrating a Cdk-dependent RLF inhibitor present in metaphase extract.
However, RLF regulation in the Xenopus system appears somewhat different from that in the other cell types. First, loss of cytoplasmic RLF during interphase in Xenopus is not dependent on Cdk activity and is not associated with an RLF inhibitor. Secondly, although relicensing of DNA is dependent on nuclear envelope permeabilization in both Xenopus and Drosophila cell-free systems (Blow and Laskey, 1988; Crevel and Cotterill, 1991), this does not seem necessary in other cell types. One attractive explanation for these apparent differences is that in Xenopus, relicensing of DNA in replicated nuclei is prevented by a Cdk-dependent inhibitor that is active only within the nucleus. Permeabilization of the nuclear envelope would then release the inhibitor and permit relicensing of the DNA by RLF-B and RLF-M.
Abbreviations used in this paper
J.J. Blow is a Lister Institute Research fellow. We thank Noel Lowndes and John Diffley for comments on the manuscript.
Received for publication 16 August 1996 and in revised form 23 October 1996.
Address all correspondence to J. Julian Blow, Imperial Cancer Research Fund, Clare Hall Laboratories, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3LD, United Kingdom. Tel.: (44) 171-269-3923. Fax: (44) 171269-3801. e-mail: email@example.com
Author notes
P. Thömmes' present address is Glaxo Wellcome Virology Unit, Gunnels Wood Road, Stevenage, Herts SG1 2NY, United Kingdom.


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Solid wood doors are made of precious species, coupled with excellent craftsmanship and durable properties, and they have a high collection value. Therefore, it is also important to do a good job of maintenance. Otherwise, it will affect the beauty of solid wood doors and reduce the service life.
Methods of maintenance
- To remove the door surface stains, a soft cotton cloth wipe should be used, Or it is easy to scratch the surface with a hard cloth. For stubborn stains, you can use neutral detergent, toothpaste, or a furniture cleaning agent. Remove the stain immediately after wiping it dry. Do not use water to rinse.
- In order to maintain the surface luster and service life of the solid wood door, furniture maintenance wax shall be used regularly, such as once every six months. When waxing, use bottled white wax and brush evenly on the surface of the solid wood door with a soft rag.
- In spring and winter, good indoor ventilation shall be maintained to keep the solid wood door in a normal temperature and humidity environment, to prevent the deformation of the solid wood door due to excessive humidity and temperature difference, and to prevent metal fittings from rusting, edge sealing, and the fall off of finishing materials. In winter, the indoor temperature should not be too high (it is better to keep it at about 20 degrees). Do not use electric heaters or other heating equipment near the wooden doors to prevent them from being heated and deformed.
- Maintaining the optimum humidity and temperature can prevent wood doors from cracking deformation. Humidification methods in addition to maintaining indoor air circulation, you can also install an indoor air humidifier or keep a few potted plants to regulate air humidity.
- Remember not to hang heavy objects on the door or let children hang on the door to play to prevent the hinges lose, the door from sagging, and shorten the service life.
- When opening or closing the door leaf, do not use too much force or open the door at too large an angle. This will not only damage the solid wood door but also hurt people with serious.
- When maintaining solid wood doors with glass, take care not to infiltrate the cleaning agent or water into the glazing bead to avoid deformation. If the glass is broken, it must be repaired by professional maintenance personnel.
- Pay attention not to rub too much at the corners of the wooden door. Otherwise, the corner paint will fall off.
- Do not often use your hands with water stains or other solutions to open the wooden door to avoid discoloration of the door lock.
- To maintain the surface gloss and service life of solid wood, doors should be regularly cleaned and dusted. And the surface can be cured with a special curing liquid for wooden decoration products.
- When painting the wall, cover the solid wood door. Avoid peeling and fading of solid wood door finishing materials caused by painting, which will affect the overall beauty.
- Solid wood doors shall not be exposed to strong sunlight for a long time, which will make the paint discolored, aging, or peeling.
Above are the solid wood door cleaning and maintenance methods and daily use attention. Hope it will be helpful to you.


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(SPOT.ph) Most of us, if not all, know a thing or two about Carlos "Botong" Fransciso. A muralist from Angono, Rizal, he was an important figure in Filipino modernism and is also one of the country's National Artists for Visual Arts. But his legacy doesn't stop there—his grandson, Carlos "Totong" Francisco II, is also a renowned artist from the Art Capital of the Philippines. If Angono is too far from you, you can get a glimpse of Totong's own artistic style through his solo show, Revival, at Robinsons Galleria in Quezon City. The show, which runs from February 1 to 15, is his first solo exhibit after a nine-year hiatus.
Totong is deeply influenced by Abstract Expressionism—the artist's response growing up in the age of Regionalism. It can be remembered that Botong, during his time, was a part of the modern art movement and broke away from Fernando Amorsolo's Romanticism. Like most artists, Totong and Botong resisted their respective status quo.
Revival is also part of Robinsons Galleria's celebration of National Arts Month and Robinsons Land Corporation's official launch of ARTablado, which is the property developer's platform to raise awareness on Filipino creativity.
Revival runs from February 1 to 15 at 3/F Veranda, Robinsons Galleria, EDSA corner Ortigas Avenue, Quezon City.


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This unique style necklace is mainly strung with white round 5-6mm pearl beads, black irregular-shaped pearl beads of 8X9mm, and some garnet pieces. Also, two balck oval faceted agate beads are used as decoration for the necklace. They are strung together with a solid steel chain. At the end of the necklace, a clasp is used as a connection. This necklace will give you every satisfaction.
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