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Welcome, Fall 2019 Students!
Welcome to Comp215 for fall 2019. At this web site, you'll find the course syllabus and schedule. Lecture slides and weekly assignments will appear on Piazza (you'll be invited to our private Piazza when classes begin). Grades will appear on Canvas. Students who are not enrolled will not be able to do the assignments or participate in the Piazza discussions.
We will soon have a SurveyMonkey signup sheet so we can know a little bit about you. That will appear here this fall.
Meanwhile, we often get questions in advance of the class from students looking for how they can better prepare themselves.
As you probably know, Comp215 will have you do all of your coding in Java, and we'll be moving to the latest Java11 this year, using the IntelliJ IDEA environment. If you're using an older, slower laptop, or if you only have a Chromebook (*), you're going to need a "real" laptop computer for Comp215. IntelliJ runs just fine on Mac, Windows, and Linux; use whatever you prefer. The exact CPU doesn't matter much either. IntelliJ does work better with at least 4GB of RAM. We recommend a minimum of 8GB of RAM and a 256GB SSD, giving you sufficient space, plenty of speed, and some robustness against you dropping your computer on the floor. Also, in the past few years several students experienced hardware failures at inopportune times. Make sure you've got good warranty coverage on your machine or you're otherwise prepared to move quickly.
Also of note, our exams will be "open Internet", which means that the world is your oyster. Make any query to Google, read any thread on StackOverflow, etc. However, we cannot guarantee a power outlet for every student in our exam rooms. You need to be confident that your laptop's battery can survive three hours of continuous usage. Battery life generally gets worse over time, so make sure your laptop is up to the task.
So what should you buy? You don't need a top-of-the-line gaming rig. Those are heavy and expensive. Instead, we recommend you get something from the "ultrabook" category. If you like Apple products, a MacBook Air or MacBook Pro will do everything you need (**). If you prefer a PC, you've got tons of choices (***).
If you learned Java in high school
If you've never written a line of Java code in your life
Don't panic! While you don't need to do anything beyond passing the prerequisite Comp classes, a couple hours of advance work over the summer will make your life a lot easier in the fall, since we hit the ground running fast. Luckily, there are some excellent and free online resources to help you from Udacity, edX, Coursera, and more. We recommend CodeAcademy. CodeAcademy's "Learn Java" tutorial is free, simple, and self-paced, and you don't have to install any software. (You can optionally pay them money for additional projects. That's not necessary.)
After you work through CodeAcademy, you might then install IntelliJ and go to the exercises below. If you want to try one of the other MOOCs, go for it, but CodeAcademy seems like the right level of detail.
While you're at it, play with the Unix command line
We never explicitly teach this in Comp215, but it's a fantastically useful thing to know. Inside every Mac is Unix, and when you run the Terminal app, you're talking directly to an old-school Unix command-line shell. It's also there from the start on every Linux machine. On Windows 10, you can install the Windows Subsystem for Linux or the minimalist MinGW, which is also part of Git for Windows. (We'll have you install Git For Windows for Comp215.) But what is the command line? How do you learn it? Why should you bother?
Once again, CodeAcademy has a free tutorial. (You can skip the paid parts.) See also the "Learn Enough" Command Line Tutorial, LinuxCommand.org, or the UNIX Tutorial for Beginners.
Feel like you're ready for Comp215? Want something a little deeper to test yourself? Here you go: two straightforward exercises and a place to look for more:
- Write a program that implements a Sieve of Eratosthenes and can print all the prime numbers less than some constant (https://en.wikipedia.org/
- Write a program that opens a plain text file and counts the number of words within (similar to the Unix "wc" program)
Beyond that, it's more important for you to work on your skills as a problem solver, regardless of the programming language. If you've got the time this summer, we recommend you try tackling the Advent of Code — 25 fun problems each year, some easy and some hard. (It's something of an annual contest that doesn't care about what programming language you use, with a leaderboard for who finishes fastest.)
If you already know Python and you're starting from scratch with Java, you might try implementing solutions to those "advent" problems first in Python then rewriting them later in Java.
Hey, why aren't we…
- Using an older version of Java? Java8, which came out in 2014, was a huge jump from prior versions, completely changing the ways you might go about solving many problems. More recently, Oracle has released Java11, which makes a number of minor improvements over Java8. Several of those improvements are useful for us.
- Using a newer version of Java? Java11 is considered a "long term supported" system by Oracle. It's going to be around for many years to come. The newer features introduced in Java12 are very minor, and the various other tools we use don't support it yet.
- Using Kotlin, Scala, C#, or some other language? Java has been around since the mid 1990's and has been hugely influential on these and other programming languages. Once you understand Java, you'll be able to rapidly learn these and many other programming languages.
- Using Eclipse or some other Java IDE? IntelliJ seems to be the best of the bunch and they're working aggressively to improve it. It's also free and available across every major platform. A version of IntelliJ is also used for Android development (in Java) and there are versions for other languages as well.
* In May 2018, Google just announced that they were supporting native Linux apps on Chromebooks, which includes the ability to run software development tools like we use in Comp215. They're only supporting this, experimentally, on the expensive Pixelbook. You shouldn't be dependent on "experimental" support for tools you'll be using seriously.
** Apple goes out of their way to keep their upcoming products a secret. Pay attention to the rumors about when Apple might announce something new. Apple has made recent updates to the MacBook Pro and MacBook Air that make them competitive with any PC laptop. You may be also able to find significant discounts on last year's machines or refurbished machines. So long as you have enough RAM and SSD storage, last year's machine may be a good choice. If you do buy an Apple, don't forget that you get a Rice discount.
*** Intel's "9th generation" chips are trickling out, resulting in some good sales on "7th" or "8th" generation machines, which still work just fine. Also, AMD processors are completely compatible with Intel and are both fast and affordable. For example, Dell offers an AMD-based Inspiron 13 7000 2-in-1 for $666 or thereabouts. If you do buy a Dell, don't forget to explore their educational discounts and to see if there are better prices at Amazon (e.g., that same Dell machine for $579). You may find similar discounts from other PC vendors.


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Welcome to Emsworth U3A
The Emsworth U3A is a friendly organisation which provides activities to promote active minds in a friendly social setting. It is affiliated to the Third Age Trust, the national body providing a wide range of services to U3As throughout the country.
New members are always welcome. For details of how to join, click Members.
Monthly Meetings
Monthly general meetings are held at 10.20a.m. on the third Friday of each month (except August and December) in St James Hall, 20 Church Path, EMSWORTH, PO10 7DP. Free tea and coffee are available from 9.30a.m.
The Speaker Secretary arranges a talk each month - Look at details of meetings in 2018 click Events.
Events and Outings
Please see Events Page for details of outings and social events that have been arranged.
Groups are a very important part of the U3A, and Emsworth U3A has a large selection of group activities. Click Groups to see a detailed list.
Emsworth U3A has an agreement with Havant U3A that we can each join each other's groups, where there is agreement within the group and available spaces. Go to Groups for more details.
© All photographs are the copyright and may not be copied or reproduced without permission

subset: commoncrawl


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[Note: This is the third part of a series explaining the department's Information Technology Transition. Ensuing articles will focus on a new Division of Motor Vehicles technology solution, IT modernization projects and the Information Technology governance model.]
A major upgrade of the department's computer aided design and drafting (CADD) system is one of the first technology programs to emerge from the new Technology Investment Plan.
Although the upgrade was included as a Legislative decision unit in 2003, it formally became part of the new process of acquiring technology solutions last year, explains project manager Ray Oldham.
The department sought and received authority from the Legislature for $475,000. The overall project cost is about $600,000, including specialized training required to prepare employees for using the software suite.
ITD has been using the design/drafting software since 1983, but hasn't upgraded the suite in six years. Oldham said ITD is about six versions behind in some applications, forcing most design consultants to downgrade their projects to ensure compatibility.
"They have to 'dummy-down' their projects so we
can work with them," he said.
Most state transportation departments and private-sector designers and consulting firms use the Bentley package, Oldham said.
When installation of the new software is completed, ITD will be compatible with nearly all of its consultants and contractors. The upgrade also will enable the department to move toward electronic bidding.
"The value is in the data, not the paper," Oldham explained. ITD will be able to utilize this data through the entire project life cycle.
ITD tries to upgrade CADD workstations every four years on a rotational basis. Two of those upgrades will be accomplished before the introduction of the new software suite.
The major upgrade will be completed in two phases, the first of which was a workflow study that included on-site interviews in each district and Headquarters and on-line screening, a technical report, a management report and implementation plan. The second phase, which began this week, includes software configuration and completion of ITD's CADD standards, followed by software installation and training.
Oldham said about 70 employees have registered for the InRoads training, which represents a radically new process. Training will be comprehensive, from start to finish. The MicroStation training assumes a good working knowledge of the software and system, which are not drastically different than previous versions. Approximately 120 employees have registered for the training.
Ten MicroStation classes and six InRoads classes already have been scheduled, along with three survey classes and two advanced InRoads classes. All training will be given on-site in the districts and at Headquarters by Bentley representatives. While training takes place, technicians will upgrade individual computers so employees can move directly from the classroom to their workstations.
Oldham worked with ITD's enterprise architects and Corporate IT to design the system upgrade and prepare it for the TIP program. Aside from a few small "bumps," the process has gone very smoothly, he said. "It actually has worked quite well. It forces you to look at everything from beginning to end."
Published 7-28-06


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Stephen McPartland Conservative, Stevenage
To ask the Secretary of State for Health, with reference to the announcement by the Prime Minister at the G7 Summit that all UK-funded clinical trials will be made openly available, what steps he is taking to require companies to publish such trials; and which diseases will be covered.
George FreemanThe Parliamentary Under-Secretary of State for Business, Innovation and Skills, The Parliamentary Under-Secretary of State for Health
The Prime Minister has written to government departments setting out his expectations on open data and transparency. Work is underway to take forward the transparency agenda nationally and internationally. This will cover diseases that are a threat to health security, for example emerging infectious diseases with pandemic potential.


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Starting tomorrow, I'll be away from home for a couple of days. My mom will undergo Cholecystectomy gallbladder surgery in a hospital in Sta. Maria Bulacan to remove her gallbladder and gallstones. It has been several months ago, when she complained that her stomach was aching. She got confined then and was immediately told that she needs to have operation. But just to make sure, we went for a 2nd opinion to a doctor here in
Now is the time. And we made sure that she's physically and mentally prepared for it. All we need to do is to keep on praying that everything will turn out well. Your prayers for my mom and our family are very much welcome.
Prevention is still better than cure, so I am sharing what I found to be the causes of gallstones to increase awareness of the things that we should avoid.
In case you have gallstones, early detection could prevent you from acquiring more severe complications. You may want to check also for its symptoms in order to make yourself better prepared.
As the saying goes, Health is Wealth. I am hoping that I'll be able to get more prayers for my mom through this... Thanks for spending time reading.Update:
My mom had undergone the traditional gallbladder surgery (cholecystectomy) instead of Laparoscopic since her gallbladder already constricted and was quite hard to remove. She stayed in the hospital for only 3 days and is now fast recovering. Thanks for all your prayers! :-)


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Word is buzzing today that 'X-Men' producer Lauren Shuler Donner would love to get Channing Tatum to play Gambit in a Wolverine-esque origin story. If you think this sounds like the best course of action, click away now. We'll wait.
This is a terrible idea. "Mon Dieu," as Gambit would say.
Yes, Tatum has been angling for the role since at least last year. But Shuler and Tatum are really confused over the point of Gambit in Marvel lore. The crux of problem? Channing Tatum is good at playing a man's man. Or a meathead. Or a jock with a heart of gold. Or a stripper with glutes you could bounce quarters off of. Everything about him screams All-American dudebro. Which is literally the opposite of what Gambit's character is.
Remy LeBeu is less Magic Mike and more Han Solo. A borderline anti-hero equipped with questionable morals and sly smile. A thief who loves women, liquor, and danger. A man who has to use his wits because the mutant powers-that-be were stingy with the steroid muscles. In a sea of spandex women and male power fantasies, Gambit is one of the few superheroes aimed almost directly at the female gaze.
So if you're going to give us our eye-candy you damn well better get the flavor right!


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It's always fun to open presents, especially when they're from Seth Godin.
Archives for December 2012
The joy of motherhood:Truth or Fiction? Part I
Could the joy of motherhood be fiction? Call me crazy, but I believe that motherhood and happiness can coexist peacefully.
Where I found a moment of joy
Listening to "Ode to Joy" made me cry during my son's last elementary school winter concert.


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Friends, there is not much more load that we can bear... the news on Lemon is as bad as it gets. Another punch to the gut for us, nothing in comparison to what he faces. I've tried to put a positive face on for you, so many truly care and it's what has been holding things together for us, but there are cracks In the veneer of my being that are growing by the minute it seems. I've spoken very little of the bureaucratic debacle we are being subjected to by government on every level including local, but it's taking a toll that is indescribable. I thought it would be therapeutic in some ways on our end while informative on your end to do videos and show certain realities. But as with absolutely everything else in my post Irma world, this too is impacted. Simple things are just not simple, will they ever be again is what I ask every day. I've enjoyed what I've been able to do, so I will try and get videos or posts up to finish out the month but I'm not sure I am able,
We have worked so hard, there has been no down time, none. It's all for the reopening, 18 hour days usually. the news about Lemon comes as a total shock in this chaos. I've been his person a very long time, and the other happy vegan loves him just as much. It's difficult to be back in this position just weeks after Princess crossed.
Lemon is very brave, he always has been. His beginning wasn't the best, we were lucky he landed with us. He's my boy, my man, and although I don't speak of him much, he's been a constant and stable presence through some of the worst times I've lived through. Hold good thoughts for him. He is comfortable, loved and supported by excellent trusted doctors. We can never know how long, but the prognosis is grim, we will do our best to maximize the time that remains and be the stewards he deserves.
Thank you friends as we continue to try and work through the shitstorm.


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I started babysitting when I was 12 yrs old. I began working with children professionally nearly 10 years ago...everything from newborns to teens, only children in the foster care system to triplets I nannied. I have ALWAYS hated when kids turned three. It is my absolute LEAST favorite age. I know I'm supposed to love my child at every age for different reasons...different developmental milestones and all that...and I do love my three year old son. Sometimes, I just want to love him from a distance...does anyone know if there are boarding schools for three year olds?
Anyway, the main reason I've always struggled with three is that at two, they're still obedient because you are (angel's voices sing "AWWW") MOM...and all the glory and fear that comes with it. At four, you can use logic most of the time to explain this is the boundary and this is why it's a boundary in our home or family. But at three you're stuck...they're no longer awe-inspired by your mom-ness but they don't care to be mature enough to listen to the logical reasons behind the various rules.
So, here we are at three. During deployment. All I really want to do sometimes is ship my son to his father but since that would be wrong...illegal...unloving...unethical...horrible...not to mention, pretty expensive since the boy is too big to fit into a flat rate box...and yes, I do love him, I won't ship him to a war-torn nation.
But seriously, I have a three year old son who is struggling with the fact that his Daddy is still far away and now his Mommy is no longer available 24/7. He loves his sister and (THANK GOD) has not yet started to view her as an enemy. He's confused. He's angry. He's sad. He's frustrated....but he's limited on how well he can express all those feelings because... he's three.
So I guess we just keep going. I keep trying to give words to his emotions, empathize with his pain but most importantly make clear and maintain the boundaries our family has always had...deployment or not...new baby or not. Eventually, I keep reminding myself, he'll be able to acknowledge his emotions and control them better because eventually he'll be four...then five...six...it goes too fast to really spend a year wishing it would go faster.


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It is important to adequately prepare key areas of your vehicle before leaving for a driving trip. Here are a few simple pre road-trip checks that one must keep in mind:
◠Check the air pressure: People often neglect checking the air pressure of the tires. Low air pressure may lead to a blowout from extra friction on highways and can decrease fuel economy too. You must check the pressure marked on the driver's side door to know the adequate tire pressure. It is important to check the pressure of all four tires and also be sure to keep a spare tire with its air pressure up-to-date.
â— Check the fluids in your car: You must safeguard your car from problems such as mechanical damage, over-heating or even a breakdown. Check the fluids like coolant and engine oil in your car on a regular basis to prevent such problems. If necessary, change the engine oil if the due date is approaching. Use high-quality oils to protect the life of your engine and to remove the additional stress on your motor.
â—The GPS: The GPS in your car should work properly to avoid losing your way. This Global Positioning System has to capability to show and guide you to get to any destination in your mind.
â—Check if the lights and signals are functional: It is necessary to sit in the car, turn on each light and see if it is working. You must also replace bulbs and fuse if it needs to be changed to avoid accident and problems while driving.
â— Battery: Make sure to check the battery of your car before your trip. You can do this visually by looking for leaks, cracks or any other damage in the battery. If the battery is more than four years old, you must try and replace it.
â—Brakes: You must ensure that the car brakes are working efficiently. Rusted brakes can cause a lot of difficulties for you. Also keep a check on the brake pads and rotors and replace them if they need to be changed.
â—Emergency equipment: Keep spare tires, an up-to-date map, flares, medical kits, fire extinguisher, flash lights and other emergency tools in the car. You never know what and when can problem arise! So be well-equipped.
â— Take a test-drive: You should test-drive your car thoroughly to check if there are any signs of trouble. If you hear unusual noises or feel the shakes in the gauges, take the car to your trusted professional service centre for a complete diagnosis of your car.
You can now follow these simple checks to make your trip a hassle free-experience.


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Pathways & interactions
Tetraspanin (IPR000301)
Short name: Tetraspanin
Overlapping homologous superfamilies
Family relationships
- Tetraspanin/Peripherin (IPR018499)
- Tetraspanin (IPR000301)
- Uroplakin-1a (IPR034765)
- Uroplakin-1b (IPR034766)
- Tetraspanin (IPR000301)
Tetraspanins are a distinct family of proteins, containing four transmembrane domains: a small outer loop (EC1), a larger outer loop (EC2), a small inner loop (IL) and short cytoplasmic tails. They contain characteristic structural features, including 4-6 conserved extracellular cysteine residues, and polar residues within transmembrane domains.
A fundamental role of tetraspanins appears to be organizing other proteins into a network of multimolecular membrane microdomains, sometimes called the `tetraspanin web'. Within this web there are primary complexes in which tetraspanins show robust, specific, and direct lateral associations with other proteins. The strong tendency of tetraspanins to associate with each other probably contributes to the assembly of a network of secondary interactions in which non-tetraspanin proteins are associated with each other via palmitoylated tetraspanins acting as linker proteins. In addition, the association of lipids, such as gangliosides and cholesterol, probably contributes to the assembly of even larger tetraspanin complexes, which have some lipid raft-like properties (e.g. resistance to solubilization in non-ionic detergents). Within the tetraspanin web, tetraspanin proteins can associate not only with integrins and other transmembrane proteins, but also with signalling enzymes such as protein kinase C and phosphatidylinositol-4 kinase. Thus, the tetraspanin web provides a mechanistic framework by which membrane protein signalling can be expanded into a lateral dimension [PMID: 12575999].
GO terms
Biological Process
No terms assigned in this category.
Molecular Function
No terms assigned in this category.
Cellular Component
GO:0016021 integral component of membrane
Contributing signatures
- PIRSF002419 (Tetraspanin)


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Where wine comes from. Wine tasting at Stoller Vineyards, Dayton, Oregon.
Archives for July 2011
Somewhat Daily Photo 7.31.11
July 31, 2011 By Leave a Comment
Somewhat Daily Photo 7.29.11
July 29, 2011 By Leave a Comment
Where I am. One of my favorite, favorite, favorite places.
Somewhat Daily Photo 7.28.11
July 28, 2011 By Leave a Comment
Phantom Ship. From this trip. It really is as blue as they say it is.
Headed For The Hills
July 28, 2011 By Leave a Comment
This weekend, we are unhooking from the grid for a whole three days, packing up the car, driving east, beyond civilization, outside cell coverage, and into the woods. With big mountains and big trees. We're going to build big fires and cook big meals. We'll breathe big gulps of mountain air and catch fish with […]
Somewhat Daily Photo 7.27.11
July 27, 2011 By 6 Comments
Desaturation. The beach is also charming when it's not sunny.


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Early childhood is a very important period of a child's development. Research has proved that the brain forms as many as 700 neural connections per second before the age of 5. Providing the right stimulus at this age that is developmentally appropriate is absolutely vital. This is what where Sesame Street Preschool endeavours to do, prepare curious little minds for a future full of possibilities.
Sesame Street Preschool is the latest initiative of Sesame Workshop in India. Sesame Workshop is the non-profit educational organization behind Sesame Street, the landmark television program that reaches millions of children every day in more than 150 countries. The Workshop's mission is to use the educational power of media to help children everywhere reach their highest potential.
Present in India since 2006, Sesame Workshop in India, produces the educational television program Galli Galli Sim Sim , an entertaining educational television show, that is broadcast on the leading children's channel POGO and the public broadcaster Doordarshan. The show is watched by over 10 million children every year. Delivered through a variety of platforms, the Workshop develops research-based content – including television programs, books, games, mobile apps and community engagement initiatives – that supports early childhood learning, helps prepare children for school, and addresses developmental needs.
This expertise is now being brought to the Indian preschool space through the Sesame Street Preschools. Sesame Street Preschools brings together the best of Sesame Street and Galli Galli Sim Sim's content, to the Indian preschool space. . Realising the need for quality preschool education, Sesame Street Preschool endeavours to change the paradigm in the preschool space making high quality education, teaching standards and learning spaces accessible to Indian children.
Here's what we offer- The Sesame Street Preschool Advantage:
- Globally acclaimed Sesame Street curriculum and content.
- A curriculum that integrates audio-visual, digital, print and the outdoors in a unique set of activities.
- A curriculum that instils the skills of Imagination, Critical Thinking, Communication and Collaboration in children.
- Specially created Sesame Street resources for educators that help them transact the Sesame Street Preschool curriculum and methodology.
- Curriculum, methodology, teacher training, policies and infrastructure benchmarked to the best in international and national early childhood education practices.
- Great emphasis on child protection and safety, empowerment of children, positive role modelling, inclusion, mutual respect and tolerance, and parent involvement.
- A 360 educational experience beyond the Sesame Street Preschools, through the Galli Galli Sim Sim television show and on digital platforms such as www.galligallisimsim.com, wwww.sesamestreet.org, Airtel VoD and games on iPad and Google play.
- Children are ready not just for school, but for life.
Says Sashwati Banerjee, Director, Sesame Street Preschool, "Sesame Street was built around a single, breakthrough insight; that if you can hold the attention of children, you can educate them. We are ushering in a new generation of global citizens with the confidence, skills and cultural awareness that they will need to thrive to become global citizens. This means nurturing children with lessons about growing happy and healthy, as well as academics. Our curriculum is based on over 40 years of insight into how children learn best and spans across six key areas of early childhood development—cognition, language, social, emotional, physical and cultural diversity. We are also in line with India's National Curriculum Framework."
Sesame Street Preschool is currently present across Alwar, Bengaluru, Bhopal, Secunderabad, Delhi, Hyderabad, Jaipur, Kanpur and Meerut, offering a preschool programme for 2 to 6 years olds. With presence beyond the schools, through the Galli Galli Sim Sim television show and the presence on the digital space through www. galligallisimsim.com and www.sesamestreet.org we aim to provide an educational experience to children, making them ready for school and for life!
Welcome to Sesame Street!
About Sesame Street Preschool:
Sesame Street Preschool is a venture of Sesame Schoolhouse, the latest educational initiative from Sesame Workshop, one of the largest non-profit informal educational organisation in the world who also produce the world acclaimed children's educational television show, Sesame Street. Sesame Workshop is present in over a 150 countries and produces educational content across television, radio, mobile, tablets and print. Sesame Workshop has been present in India since 2006, through its television show, Galli Galli Sim Sim.
In our complex and rapidly changing world, young learners need an energetic head start to succeed. That's what Sesame Street Preschool is all about. We're setting a new standard for early childhood education through our unique preschool and afterschool programmes. It is a learning experience like no other, brought to life by the fun and furry characters of Sesame Street and its Indian adaptation Galli Galli Sim Sim, India's first educational program just for children. Our unique brand of age-appropriate education puts kids at the centre of everything, from playful project-based activities to an environment made for rampant imagination. And the results are dramatic: children who attend are better-rounded, better adjusted, and more confident. In short, they're better equipped for school and life.
Our Preschool and afterschool programmes are developed by the non-profit educational organization behind Sesame Street and based on 40 years of insight into how children learn best. Sesame Schoolhouse is the latest milestone in Sesame Street's mission: to empower children everywhere to reach their highest potential.
To know more please visit www.sesameschoolhouse.in, or follow us on: facebook / linkedin. For any queries please contact:
Shobha Kapoor
Email- firstname.lastname@example.com
Mobile- +91- 9811734703


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I love waking up next to my husband.
I can't believe it's December and I am still running outside. And on a beautiful sunny day in the 60's! I never thought I would even make it into November! Just wait until you see me running in the snow.
Thankful that I get to run by a beautiful river whenever I want to! My favorite part of this run!
Sometimes I say things I don't really mean out of exaggeration. (I think there should be two a's in that word just to exaaggerate.)
People should smile at each other more, it would make everyone happier.
I love seeing people out walking their dogs.
I am glad that I can eat real food again instead of crackers all day, although my saltine diet was making me skinny, or maybe that was the flu.
So lucky I am hearing one of my favorite Christmas tunes resonating through the apartment (sung by my most wonderful Hubbkin)…
I love Christmastime.


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By Richard Heap, Richard Heap Richard Heap 21 December 2007
Each week we meet a property character from a different age bracket. This week Richard Heap meets Rupert Johnson, 45, a tennis player and office secret Santa enthusiast
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New Research In
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- Systems Biology
Historical contingency and the evolution of a key innovation in an experimental population of Escherichia coli
Contributed by Richard E. Lenski, April 9, 2008 (received for review March 26, 2008)
The role of historical contingency in evolution has been much debated, but rarely tested. Twelve initially identical populations of Escherichia coli were founded in 1988 to investigate this issue. They have since evolved in a glucose-limited medium that also contains citrate, which E. coli cannot use as a carbon source under oxic conditions. No population evolved the capacity to exploit citrate for >30,000 generations, although each population tested billions of mutations. A citrate-using (Cit+) variant finally evolved in one population by 31,500 generations, causing an increase in population size and diversity. The long-delayed and unique evolution of this function might indicate the involvement of some extremely rare mutation. Alternately, it may involve an ordinary mutation, but one whose physical occurrence or phenotypic expression is contingent on prior mutations in that population. We tested these hypotheses in experiments that "replayed" evolution from different points in that population's history. We observed no Cit+ mutants among 8.4 × 1012 ancestral cells, nor among 9 × 1012 cells from 60 clones sampled in the first 15,000 generations. However, we observed a significantly greater tendency for later clones to evolve Cit+, indicating that some potentiating mutation arose by 20,000 generations. This potentiating change increased the mutation rate to Cit+ but did not cause generalized hypermutability. Thus, the evolution of this phenotype was contingent on the particular history of that population. More generally, we suggest that historical contingency is especially important when it facilitates the evolution of key innovations that are not easily evolved by gradual, cumulative selection.
At its core, evolution involves a profound tension between random and deterministic processes. Natural selection works systematically to adapt populations to their prevailing environments. However, selection requires heritable variation generated by random mutation, and even beneficial mutations may be lost by random drift. Moreover, random and deterministic processes become intertwined over time such that future alternatives may be contingent on the prior history of an evolving population. For example, multiple beneficial mutations will arise in some unpredictable order (1, 2), and those that are substituted first may differ from others in their pleiotropic effects and epistatic interactions (3, 4), thus constraining some evolutionary paths while potentiating other outcomes (5⇓⇓⇓–9). These accidents of history may even determine the survival or extinction of entire lineages, given the capricious and sudden nature of some environmental changes (10⇓–12).
Stephen Jay Gould maintained that these historical contingencies make evolution largely unpredictable. Although each change on an evolutionary path has some causal relation to the circumstances in which it arose, outcomes must eventually depend on the details of long chains of antecedent states, small changes in which may have enormous long-term repercussions (13⇓–15). Thus, Gould argued that contingency renders evolution fundamentally quirky and unpredictable, and he famously suggested that replaying the "tape of life" from some point in the distant past would yield a living world far different from the one we see today. Simon Conway Morris countered that natural selection constrains organisms to a relatively few highly adaptive options, so that "the evolutionary routes are many, but the destinations are limited" (16). He and others point to numerous examples of convergent evolution as evidence that selection finds the same adaptations despite the vagaries of history. Evolution may thus be broadly repeatable, and multiple replays would reveal striking similarities in important features, with contingency mostly confined to minor details (16⇓⇓–19).
Of course, replaying life's tape on the planetary scale is impossible, but careful experiments can examine the role of contingency in evolution on a more modest scale (15, 20, 21). To address the repeatability of evolutionary trajectories and outcomes, the long-term evolution experiment (LTEE) with Escherichia coli was started in 1988 with the founding of 12 populations from the same clone (2). These populations were initially identical except for a neutral marker that distinguished six lines from six others. They have since been propagated by daily 1:100 serial transfer in DM25, a minimal medium containing 25 mg/liter glucose as the limiting resource (2, 22). Environmental conditions have been controlled, constant, and identical for all 12 lines. To date, each population has evolved for >44,000 generations, and samples have been frozen every 500 generations, providing a rich "fossil record" (23). Moreover, these samples remain viable, allowing us to perform simultaneous measurements and other experiments with bacteria from different generations. The founding strain is strictly asexual, and thus populations have evolved by natural selection and genetic drift acting on variation generated solely by spontaneous mutations that occurred during the experiment. Thus, the LTEE allows us to examine the effects of contingency that are inherent to the core evolutionary processes of mutation, selection, and drift.
Previous analyses of this experiment have shown numerous examples of parallel phenotypic and genetic evolution. All twelve populations underwent rapid improvement in fitness that decelerated over time (2, 3, 22, 23). All evolved higher maximum growth rates on glucose, shorter lag phases upon transfer into fresh medium, reduced peak population densities, and larger average cell sizes relative to their ancestor (22⇓⇓⇓–26). Ten populations evolved increased DNA supercoiling (27), and those populations examined to date show parallel changes in global gene-expression profiles (4, 28, 29). At least three genes have substitutions in all 12 populations (30, 31), and several others have substitutions in many populations (27⇓⇓–30), even though most loci harbor no substitutions in any of them (32). At the same time, there has also been some divergence between populations. Four have evolved defects in DNA repair, causing mutator phenotypes (3, 33). There is subtle, but significant, between-population variation in mean fitness in the glucose-limited medium in which they evolved (2, 23). In media containing other carbon sources, such as maltose or lactose, the variation in performance is much greater (34). And while the same genes often harbor substitutions, the precise location and details of the mutations almost always differ between the populations (27⇓⇓⇓–31).
Throughout the duration of the LTEE, there has existed an ecological opportunity in the form of an abundant, but unused, resource. DM25 medium contains not only glucose, but also citrate at a high concentration. The inability to use citrate as an energy source under oxic conditions has long been a defining characteristic of E. coli as a species (35, 36). Nevertheless, E. coli is not wholly indifferent to citrate. It uses a ferric dicitrate transport system for iron acquisition, although citrate does not enter the cell in this process (37, 38). It also has a complete tricarboxylic acid cycle, and can thus metabolize citrate internally during aerobic growth on other substrates (39). E. coli is able to ferment citrate under anoxic conditions if a cosubstrate is available for reducing power (40). The only known barrier to aerobic growth on citrate is its inability to transport citrate under oxic conditions (41⇓–43). Indeed, atypical E. coli that grow aerobically on citrate (Cit+) have been isolated from agricultural and clinical settings, and were found to harbor plasmids, presumably acquired from other species, that encode citrate transporters (44, 45).
Other findings suggest that E. coli has the potential to evolve a Cit+ phenotype. Hall (41) reported the only documented case of a spontaneous Cit+ mutant in E. coli. He hypothesized that some complex mutation, or multiple mutations, activated cryptic genes that jointly expressed a citrate transporter, although the genes were not identified. Pos et al. (43) identified an operon in E. coli K-12 that apparently allows anaerobic citrate fermentation, and which includes a gene, citT, encoding a citrate–succinate antiporter. High-level constitutive expression of this gene on a multicopy plasmid allows aerobic growth on citrate, but the native operon has a single copy that is presumably induced only under anoxic conditions.
Despite this potential, none of the 12 LTEE populations evolved the capacity to use the citrate that was present in their environment for over 30,000 generations. During that time, each population experienced billions of mutations (22), far more than the number of possible point mutations in the ≈4.6-million-bp genome. This ratio implies, to a first approximation, that each population tried every typical one-step mutation many times. It must be difficult, therefore, to evolve the Cit+ phenotype, despite the ecological opportunity. Here we report that a Cit+ variant finally evolved in one population by 31,500 generations, and its descendants later rose to numerical dominance. The new Cit+ function has been the most profound adaptation observed during the LTEE and has had major consequences. As we will show, the population achieved a severalfold increase in size. Moreover, a stable polymorphism emerged, with a Cit− minority coexisting with the new Cit+ majority. Interestingly, the population that evolved the Cit+ function is not one that had previously become hypermutable. It is also intriguing that this key innovation evolved so late in the experiment, given that the rate of fitness improvement had declined substantially in all of the populations (3, 23).
The long-delayed and unique evolution of the Cit+ phenotype might indicate that it required some unusually rare mutation, such as a particular chromosomal inversion, that does not scale with typical mutation rates. Alternatively, the occurrence or phenotypic expression of the mutation that generated the Cit+ function might depend on one or more earlier mutations, such that its evolution was contingent on the particular history of that population. Contingent adaptations should tend to be complex and require multiple steps, some of which might not be beneficial, at least not uniquely so given other advantageous paths. Otherwise, cumulative selection would predictably favor the same steps, and the evolutionary path should be repeatable (18). Contingent adaptations should thus display two characteristics. First, independent origins should be rare, because the same historical sequences would rarely recur (19). Second, significant time-lags should occur between the presentation of ecological opportunities or challenges and the evolution of those traits that confer adaptation to those circumstances (46).
Can the hypothesis of contingent adaptation be rigorously tested in the case of the evolution of the Cit+ function? And can this hypothesis be formally distinguished from the alternative explanation that this new function required an unusually rare mutation, but one that was not historically contingent? The answer to both questions is yes, owing to certain features of bacteria in general and the LTEE in particular that allow us quite literally to replay evolution from various points in a population's history. We will first describe the emergence of this new function, and then present our experiments to distinguish between the hypotheses of mutational rarity and historical contingency in the origin of this key innovation.
Evolution of Cit+ Function in Population Ara-3.
The LTEE populations are transferred daily into fresh medium, and the turbidity of each is checked visually at that time. Owing to the low concentration of glucose in DM25 medium, the cultures are only slightly turbid when transferred. Occasional contaminants that grow on citrate have been seen over the 20 years of this experiment. These contaminated cultures reach much higher turbidity owing to the high concentration of citrate in the medium, which allows the contaminants to reach high density. (When contamination occurs, the affected population is restarted from the latest frozen sample.) After ≈33,127 generations, one population, designated Ara-3, displayed significantly elevated turbidity that continued to rise for several days (Fig. 1). A number of Cit+ clones were isolated from the population and checked for phenotypic markers characteristic of the ancestral E. coli strain used to start the LTEE: all were Ara−, T5-sensitive, and T6-resistant, as expected (2). DNA sequencing also showed that Cit+ clones have the same mutations in the pykF and nadR genes as do clones from earlier generations of the Ara-3 population, and each of these mutations distinguishes this population from all of the others (30). Therefore, the Cit+ variant arose within the LTEE and is not a contaminant.
The evolved Cit+ variant grows to high density in DM0 (a citrate-only medium), produces vigorous colonies on minimal citrate (MC) agar plates, and causes a positive color change on Simmon's citrate agar, all of which indicate that it can use citrate as a sole carbon source. In DM25, Cit+ cells undergo a period of rapid growth on glucose that is followed by slower growth on citrate (Fig. 2). Also, growth on citrate is inhibited by the citrate analog 5-fluorocitrate (data not shown), as was observed for the one previously reported Cit+ mutant of E. coli (42, 43).
Cit+ clones could be readily isolated from the frozen sample of population Ara-3 taken at generation 33,000. To estimate the time of origin of the Cit+ trait, we screened 1,280 clones randomly chosen from generations 30,000, 30,500, 31,000, 31,500, 32,000, 32,500, and 33,000 for the capacity to produce a positive reaction on Christensen's citrate agar, which provides a sensitive means to detect even weakly citrate-using cells. No Cit+ cells were found in the samples taken at 30,000, 30,500, or 31,000 generations. Cit+ cells constituted ≈0.5% of the population at generation 31,500, then 15% and 19% in the next two samples, but only ≈1.1% at generation 33,000. It appears that the first Cit+ variant emerged between 31,000 and 31,500 generations, although we cannot exclude an earlier origin. The precipitous decline in the frequency of Cit+ cells just before the massive population expansion suggests clonal interference (47), whereby the Cit− subpopulation produced a beneficial mutant that out-competed the emerging Cit+ subpopulation until the latter evolved some other beneficial mutation that finally ensured its persistence. The hypothesis of clonal interference implies that the early Cit+ cells were very poor at using citrate, such that a mutation that improved competition for glucose could have provided a greater advantage than did marginal exploitation of the unused citrate.
Indeed, the Cit+ clones isolated from generations 32,500 and earlier grow much more slowly on citrate than those from 33,000 generations and later. After depleting the glucose in DM25, the earliest Cit+ clones grow almost imperceptibly, if at all, for many hours before they begin efficiently using the citrate (data not shown), whereas later Cit+ clones switch to growth on citrate almost immediately (Fig. 2). Thus, the population expansion between generations 33,000 and 33,500 (Fig. 1) was triggered by one or more mutations that improved citrate utilization, rather than by the growth of the original Cit+ mutant. This finding also raises the question of whether weak Cit+ mutants might exist in any other LTEE population. We therefore screened the other 11 populations, in most cases by using samples from generation 41,500. None of 220 cultures inoculated with heterogeneous population samples grew in glucose-free DM0, nor did any of >3,500 clones show a positive reaction on Christensen's citrate agar.
Historical Contingency in the Evolution of Cit+.
We performed three experiments to test whether the evolution of the Cit+ function required an unusually rare mutation or, alternatively, was historically contingent and depended on the prior evolution of a certain genetic background. All three experiments used clones sampled from many generations of population Ara-3 to replay evolution starting from different genetic backgrounds. The two hypotheses make distinct predictions about the propensity of these backgrounds to re-evolve the Cit+ phenotype (Fig. 3). According to the rare-mutation hypothesis, Cit+ variants should evolve at the same low rate regardless of the generation of origin of the clone with which a replay started. By contrast, the historical-contingency hypothesis predicts that the mutation rate to Cit+ should increase after some potentiating genetic background has evolved. Thus, Cit+ variants should re-evolve more often in the replays using clones sampled from later generations of the Ara-3 population.
In our first experiment, we performed the replays under the same conditions as the LTEE. We isolated three random clones from each of twelve time points, from the ancestor to 32,500 generations (Table 1), and obtained neutral Ara+ mutants of each clone to embed as protection against accidental cross-contamination during the experiment (2). In total, there were 72 replay populations, six from each generation, each founded by a single clone. These populations evolved for ≈3,700 generations, and they were checked visually each day for the elevated turbidity indicative of the Cit+ phenotype. We also tested samples on MC and Christensen's citrate agar plates every 250 generations, with incubation for up to a week. New Cit+ variants evolved in four replay populations, all founded by clones from later generations of the original population (Table 1). These Cit+ variants emerged between 750 and 3,700 generations of the replay experiment.
Our second experiment also looked for Cit+ mutants derived from clones sampled at various times in the history of population Ara-3. This time, however, we incubated large populations of cells on MC plates, enabling us to test more clones and more cells of each clone. We also allowed a long incubation time to facilitate the growth and detection of very weak Cit+ mutants, as well as mutations that might occur as cells sat starving on the plates. In this experiment, ≈3.9 × 108 cells of each of the same 68 clones used in the first replay experiment were spread on each of five MC plates, and these 340 plates were then incubated for 59 days. Five plates produced Cit+ mutants, and all used clones from generations 32,000 or 32,500 of the original population (Table 1). None of the particular clones that evolved Cit+ in this experiment did so in the first one, although there was overlap in the generations from which those clones were sampled.
The third replay experiment was similar in design to the second, but on a larger scale. We isolated 20 clones from each of 13 time points in the history of population Ara-3, again through 32,500 generations. We generated and tested 10 replicate cultures of each evolved clone and 200 replicates of the ancestor. Each culture grew to ≈1–2 × 1010 cells, which were pelleted by centrifugation, spread on an MC plate, and incubated for 45 days. In total, ≈4.0 × 1013 cells were tested for their ability to use citrate in this experiment. Cit+ mutants emerged from eight cultures representing seven clones (Table 1), with one clone yielding mutants in two replicate cultures. Four clones that produced Cit+ mutants came from generations 31,000 and later, two were from generation 27,000, and one (the one that produced two mutants) was from generation 20,000. We found no Cit+ mutants among any of the 200 ancestral cultures, nor among any of the other 600 cultures that used clones isolated before generation 20,000.
Interestingly, 7 of the 8 plates that yielded a mutant Cit+ colony produced multiple colonies, including one with 137 colonies. This pattern illustrates the "jackpot" effect discovered by Luria and Delbrück (48), and it implies that mutations arose during the population growth before plating on MC agar. On the other hand, the Cit+ colonies were not observed until at least 8 days of incubation and, in one case, they were first seen after 28 days. These late appearances suggest that the mutations to Cit+ occurred after plating. One possible explanation for this apparent discrepancy is that the mutants grow very slowly but, in fact, they typically produce visible colonies in only 2 days when retested on MC plates. Another potential explanation is that the high density of Cit− cells on the plates interfered with the growth and detection of emerging Cit+ colonies. To test this possibility, we seeded dense Cit− cultures with a few Cit+ cells before plating on MC agar, but the Cit+ colonies were seen after only 2–3 days. The rapid growth of Cit+ colonies occurred even when the Cit+ cells had grown on glucose, and not on citrate, before plating. These results imply that mutations to Cit+ occurred after cultures were plated on MC agar. This conclusion, taken together with the jackpot distribution of Cit+ mutants, indicates that the phenotypic change required two mutations, one of which occurred during the culture growth before plating and the other after plating.
Statistical Analysis of the Replay Experiments.
All three experiments show the same tendency for Cit+ variants to evolve more often from clones sampled in later than earlier generations of population Ara-3 (Table 1). To calculate the significance of these data, we performed Monte Carlo resampling tests (shuffling without replacement) by using the Statistics101 Re-sampling Simulator version 1.0.6 (www.statistics101.net). For each experiment, we compared the observed mean generation of those clones that yielded Cit+ variants to the mean expected under the null hypothesis that clones from all generations have equal likelihood. The null thus corresponds to the rare-mutation hypothesis laid out in the Introduction. We ran one million resampling iterations for each experiment. The deviations from the null expectations range from marginally to highly significant in the three experiments, and in all cases they support the historical-contingency hypothesis, according to which clones from later generations have greater propensity to evolve the Cit+ phenotype (Table 2). Although the third experiment was the largest, it was the least significant, owing primarily to the production of two Cit+ mutants by a 20,000-generation clone. We also used the Z-transformation method (49) to combine the probabilities from our three experiments, and the result is extremely significant (P < 0.0001) whether or not the experiments are weighted by the number of independent Cit+ mutants observed in each one. Furthermore, the potentiation effect in later generations is underestimated by these tests, because the number of cells trended lower in later-generation cultures owing to the evolution of larger cells that reach lower population density (24, 25, 41, 50).
These analyses compel us to reject the hypothesis that a rare mutation could have produced a Cit+ variant with equal probability at any point in the LTEE. Some unusually rare mutation might be involved, but its rarity does not provide a sufficient explanation for the unique and exceptionally slow evolution of this new function during the LTEE. Our results instead support the hypothesis of historical contingency, in which a genetic background arose that had an increased potential to evolve the Cit+ phenotype.
Rates of Mutation to Cit+ and Quantification of the Potentiation Effect.
Given the fraction of cultures that produce no mutants (P0) and the number of cells per culture (N), one can estimate the mutation rate as μ = −(ln P0)/N. The corresponding 95% confidence interval (CI) can also be calculated from the Poisson distribution based on the uncertainty in P0, which is much greater than the uncertainty in N. By comparing the mutation rate to Cit+ between clones, one can in principle quantify the potentiation effect in the evolved genetic background. However, we cannot unambiguously identify which evolved clones remain nonpotentiated owing to the very low mutation rate to Cit+ even in the potentiated clones. We also cannot separate potentiated and nonpotentiated clones by generation because some generational samples may be polymorphic.
We therefore performed an independent series of fluctuation tests using seven clones that yielded Cit+ mutants in at least one replay experiment. These additional tests permit an unbiased estimate of the mutation rate to Cit+ in the potentiated background. We prepared 40 replicate cultures for each potentiated clone, and 280 for the ancestor. Potentiated and ancestral cultures had, on average, 1.1 and 1.5 × 1010 cells, respectively, which were harvested and incubated on MC agar plates for 45 days.
None of the ancestral cultures yielded any Cit+ mutants. We can nonetheless calculate an upper limit to the mutation rate by noting that the Poisson distribution has a 5% probability of yielding zero events when the expectation is three. With no more than three mutations among the 8.4 × 1012 cells tested here and in the third replay experiment, the upper bound on the ancestral mutation rate to Cit+ is 3.6 × 10−13 per cell per generation (Fig. 4). To the best of our knowledge, this value is the lowest upper bound ever reported for a mutation rate that has been experimentally measured. It is also probably far too high because no mutations were actually observed for the ancestor, nor were any found among another 9.0 × 1012 cells of 60 clones sampled through 15,000 generations; and because some cell turnover and other DNA activity probably occurred during the many days that plates were incubated.
Even among the potentiated clones, the rate of mutation to Cit+ is extremely low. Cit+ mutants arose in 2 of the 280 new cultures, giving an estimate of 6.6 × 10−13 for the mutation rate, with the 95% CI extending from 7.9 × 10−14 to 2.4 × 10−12 (Fig. 4). (Although the upper bound of the CI for the ancestor overlaps the lower bound of the CI for the potentiated clones, that upper bound does not overlap the point estimate for the potentiated clones, indicating a significant difference and adding further support to the replay experiments.) The potentiated genetic background thus increases the mutation rate to Cit+ at least 2-fold, and probably much more. However, even this potentiated value represents an unusually low mutation rate. A typical mutation rate in E. coli is ≈5 × 10−10 per base pair per generation (51). Such a low rate suggests that the final mutation to Cit+ is not a point mutation but instead involves some rarer class of mutation or perhaps multiple mutations. The possibility of multiple mutations is especially relevant, given our evidence that the emergence of Cit+ colonies on MC plates involved events both during the growth of cultures before plating and during prolonged incubation on the plates.
Another issue is whether the potentiating effect might indicate the evolution of a generalized hypermutability in population Ara-3. Previous surveys of mutation rates in the 12 LTEE lines found that 4 had become mutators by generation 20,000, although population Ara-3 retained the low ancestral mutability (3, 33). To investigate this issue further, we performed another series of fluctuation tests using the same seven potentiated clones to determine their rate of mutation from Ara− to Ara+, which serves as a proxy for the background mutation rate (33). This mutation reverts a phenotype that was knocked out during the derivation of a predecessor to the ancestor of the LTEE (2, 52). Sequence analysis indicates that most Ara+ mutants have a mutation from GAC to GGC at codon 92 of the araA gene, restoring that codon to its distant ancestral state. Forty cultures of each clone were grown in DM1000 (1 g of glucose per liter), and the cells were spread on minimal agar plates containing arabinose. At the same time, 280 cultures of the ancestor were tested in the same way. The ancestral mutation rate to Ara+ is 2.3 × 10−10 per cell per generation calculated by using the P0 method (95% CI from 1.8 × 10−10 to 2.9 × 10−10), a typical rate for point mutations. The rate for potentiated clones is also 2.3 × 10−10 (95% CI from 2.0 × 10−10 to 2.7 × 10−10); in fact, these estimates are within 1% of each other (Fig. 4). We conclude that general hypermutability is not responsible for the elevated mutation rate to Cit+ in the potentiated clones.
Frequency-Dependent Selection Maintains Ecological Diversity.
To this point, we have examined the evolutionary origin of the Cit+ function in population Ara-3. We now turn to its ecological consequence. The Cit+ phenotype did not achieve fixation during the population expansion but, instead, Cit− cells persisted as a minority. When we mixed Cit− and Cit+ clones at different initial frequencies, they stably coexisted over many serial transfers (Fig. 5). In these mixtures, the Cit− cells gradually approached an equilibrium frequency of roughly 1%, regardless of their initial frequency. We saw a transient jump in the frequency of the Cit− subpopulation on days 7 and 8, which was probably caused by accidentally using a glucose-only medium on those days. After that perturbation, the populations resumed their previous trajectories. Negative frequency-dependent selection thus maintains the polymorphism.
This stable coexistence suggests that the Cit− cells are superior to the Cit+ cells in competition for glucose, allowing the former to persist as glucose specialists. Indeed, the Cit− cells have a shorter lag phase and higher growth rate on glucose than do the Cit+ cells (Fig. 2). These differences were also evident when we monitored the intraday dynamics of mixtures of Cit− and Cit+ cells (data not shown).
Discussion and Future Directions
E. coli cells cannot grow on citrate under oxic conditions, and that inability has long been viewed as a defining characteristic of this important, diverse, and widespread species. In a long-term experiment, we propagated 12 populations of E. coli, all founded from the same ancestral strain, in a medium containing glucose, which is the limiting resource, and abundant citrate. For more than 30,000 generations, none of them evolved the capacity to use the citrate, although billions of mutations occurred in each population, such that any typical base pair mutation would have been tested many times in each one. It is clearly very difficult for E. coli to evolve this function. In fact, the mutation rate of the ancestral strain from Cit− to Cit+ is immeasurably low; even the upper bound is 3.6 × 10−13 per cell generation, which is three orders of magnitude below the typical base pair mutation rate. Nevertheless, one population eventually evolved the Cit+ function, whereas all of the others remain Cit− after more than 40,000 generations.
We demonstrated that the evolution of this new function was contingent on the history of the population in which it arose. In particular, we showed that one or more earlier mutations potentiated the evolution of this function by increasing the mutation rate to Cit+, although even the elevated rate is much lower than a typical mutation rate. The potentiated cells are not generally hypermutable. Rather, their potentiation appears to be specific to the Cit+ function, which suggests two possible mechanisms. One mechanism is epistasis, whereby the functional expression of the mutation that finally yielded the Cit+ phenotype requires interaction with one or more mutations that evolved earlier. A second possibility is that the physical production of the mutation that produced the Cit+ phenotype requires some previous mutation that allows the final sequence to be generated. For example, the insertion of a mobile genetic element creates new sequences at its junctures, and one of these new sequences might then undergo a mutation that generates a final sequence that could not have occurred without the insertion. The E. coli genome has many insertion-sequence elements (53), some of which have been active in the LTEE (54⇓–56). Whatever the mechanism, this potentiation made the Cit+ function mutationally accessible, and a weak Cit+ variant emerged by 31,500 generations.
The origin of the Cit+ function also had profound consequences for the ecology and subsequent evolution of that population. This new capacity was refined over the next 2,000 generations, leading to a massive population expansion as the Cit+ cells evolved to exploit more efficiently the abundant citrate in their environment. Although the Cit+ cells continued to use glucose, they did not drive the Cit− subpopulation extinct because the Cit− cells were superior competitors for glucose. Thus, the overall diversity increased as one population gave rise evolutionarily to an ecological community with two members, one a resource specialist and the other a generalist.
The evolution of the new Cit+ function represents a key innovation that involves multiple steps, and it provides an explicit demonstration of the importance of historical contingency in evolution. It also transcends the phenotypic boundaries of a diverse and well studied species, and led to an ecological transition from a single population to a two-member community. Our future research on this fascinating case of evolution in action will revolve around four themes: genetics, physiology, ecology, and speciation.
What is the genetic basis of this evolutionary innovation? The emergence of the Cit+ phenotype in population Ara-3 indicates at least two important genetic events: the origin of the function in its weak form, and its subsequent refinement for efficient use of citrate. The replay experiments indicate an even more complex picture that must involve, at a minimum, three important genetic events. At least one mutation in the LTEE was necessary to produce a genetic background with the potential to generate Cit+ variants, while the distribution and dynamics of Cit+ mutants in fluctuation tests indicate at least two additional mutations are involved. To find the relevant mutations, we will perform whole-genome resequencing, which has become a powerful approach that is well suited to experimental evolution (57⇓–59). We expect to find dozens of mutations relative to the ancestor (22), which will complicate identification of those changes that were important specifically for the origin of the Cit+ function. However, some of the key changes should become apparent if we also resequence a Cit− clone from the same population around the time that the Cit+ variants first emerged. Once candidate genes and mutations have been identified, we can examine the other 19 Cit+ variants from our replay and mutation-rate experiments for parallel changes.
We are especially eager to find the potentiating mutation or mutations. We want to know whether the potentiating mutation interacts epistatically with a later mutation to allow expression of the Cit+ function or, alternatively, whether it was physically required for the later mutation to occur. We also want to test whether the potentiating mutation was itself beneficial or, alternatively, a neutral or deleterious change that fortuitously hitchhiked to high frequency. We anticipate that identifying the potentiating mutation will be especially challenging, however, because its only known phenotype is to increase the rate of production of certain mutants that are themselves extremely rare.
Once we have identified all of the relevant mutations, it might be interesting to model the population dynamics that govern the emergence of this new function. Such a model would require not only all of the relevant mutation rates but also the ecological phenotypes of the mutants, including their growth rates on glucose and citrate as well as their abilities to transition between the two resources. A satisfactory model should also reflect the stochastic origin of mutations, the role of random drift, and the possibility of alternative mutational paths to the phenotype of interest (9, 60).
What physiological mechanism has evolved that allows aerobic growth on citrate? E. coli should be able to use citrate as an energy source after it enters the cell, but it lacks a citrate transporter that functions in an oxygen-rich environment. One possibility is that the Cit+ lineage activated a "cryptic" transporter (41), that is, some once-functional gene that has been silenced by mutation accumulation. This explanation seems unlikely to us because the Cit− phenotype is characteristic of the entire species, one that is very diverse and therefore very old. We would expect a cryptic gene to be degraded beyond recovery after millions of years of disuse. A more likely possibility, in our view, is that an existing transporter has been coopted for citrate transport under oxic conditions. This transporter may previously have transported citrate under anoxic conditions (43) or, alternatively, it may have transported another substrate in the presence of oxygen. The evolved changes might involve gene regulation, protein structure, or both (61).
What will be the long-term fates of the coexisting Cit− and Cit+ subpopulations? We showed they stably coexist owing to the inferiority of the Cit+ cells in competition for glucose. However, the Cit+ lineage might eventually acquire mutations that compensate for its inferior performance on glucose, thus undermining the coexistence. Compensation for maladaptive side-effects of adaptations, including resistance to phages and antibiotics, has been observed in many other experiments with bacteria (62⇓⇓⇓–66). Moreover, the Cit+ subpopulation is much larger than the Cit− subpopulation, so it should experience more beneficial mutations even without compensation. On the other hand, coexistence would be strengthened if selection in the Cit+ subpopulation favors specialization on citrate. In the same way that we established multiple populations for retrospective replays in this study, we can establish multiple communities to examine their prospective evolution. We can also vary environmental factors, such as the presence or absence of glucose, and the presence or absence of the Cit− subpopulation, to investigate how they influence the future evolution of the Cit+ lineage.
Will the Cit+ and Cit− lineages eventually become distinct species? According to the biological species concept widely used for animals and plants, species are recognized by reproductive continuity within species and reproductive barriers leading to genetic isolation between species (67). Although the bacteria in the LTEE are strictly asexual, we can nonetheless imagine testing this criterion by producing recombinant genotypes. In particular, we could move mutations that are substituted in the evolving Cit+ lineage into a Cit− background to test whether they reduce fitness in their ancestral context. One could also perform the reciprocal experiment, although we anticipate more rapid evolution in the Cit+ lineage because it has acquired a key innovation that substantially changed its ecological niche. Such experiments would require, of course, controls to examine the fitness effects of the same mutations in the lineage where they arose. If the Cit+ lineage is indeed evolving into a new species, then we expect, with time, that more and more of the beneficial mutations substituted in that lineage would be detrimental in the ecological and genetic context of its Cit− progenitor.
In any case, our study shows that historical contingency can have a profound and lasting impact under the simplest, and thus most stringent, conditions in which initially identical populations evolve in identical environments. Even from so simple a beginning, small happenstances of history may lead populations along different evolutionary paths. A potentiated cell took the one less traveled by, and that has made all the difference.
Materials and Methods
The Long-term Evolution Experiment.
The LTEE is described in detail elsewhere (2, 22). Briefly, two ancestral clones of E. coli B were each used to found six populations. The ancestors differ by a single mutation that allows one of them to use arabinose (Ara+). Ara− and Ara+ cells make red and white colonies, respectively, on tetrazolium–arabinose (TA) plates, but the mutation is neutral in the environment of the LTEE (2). The twelve populations have been propagated for almost 20 years by daily serial dilution in DM25, a minimal salts medium that has 139 μM glucose and 1,700 μM citrate (2). Given 1:100 dilution and regrowth, the populations achieve ≈6.64 generations per day, and they have evolved for over 40,000 generations in this experiment to date. Every 500 generations, population samples are frozen at −80°C with glycerol added as a cryoprotectant. These samples contain all of the diversity present in a population at that generation. Except as otherwise noted, all bacteria used in this study come from samples of population Ara-3.
Media and Culture Conditions.
DM0 is identical to DM25 except that it contains no glucose. MC plates have the same formulation as liquid DM0, except that agar is added to solidify the medium and the concentration of citrate is higher to allow large colonies. Before starting an experiment, bacteria were preconditioned in the relevant experimental medium unless otherwise stated. For additional details, see supporting information (SI) Materials and Methods.
Analysis of the Population Expansion.
When the increased turbidity was observed in population Ara-3, we froze samples of the two previous days' cultures, which had been temporarily stored at 4°C. We also froze samples after every transfer for several weeks. We later revived these and other samples taken between generations 30,000 and 35,000 to quantify the population expansion. For details, see SI Materials and Methods.
Searches for Cit+ Variants.
To determine when the earliest Cit+ variant arose in population Ara-3, we randomly chose 1,280 clones from samples frozen at generations 30,000, 30,500, 31,000, 31,500, 32,000, 32,500, and 33,000. We transferred colonies to Christensen's citrate agar plates, which were incubated for 10 days and examined daily for evidence of positive reactions. All putative Cit+ clones were retested on Christensen's agar for confirmation, and a subset was tested on MC agar. All clones that had positive reactions on Christensen's plates also grew on MC plates. To find Cit+ cells that might be present at low frequency in the other 11 LTEE populations, we tested their genetically heterogeneous full-population samples for growth in DM0. We allowed the top of each sample to thaw and inoculated 50 μl into LB broth. After overnight growth, five 10-ml DM0 cultures were inoculated with 100 μl of 100-fold dilutions of each LB culture. The DM0 cultures were incubated for 28 days with periodic checks for turbidity.
Growth Curves.
Three representative Cit− clones and three representative Cit+ clones from generation 33,000 were separately preconditioned in DM25. The Cit− clones were then combined, as were the Cit+ clones, and the mixtures were diluted in DM25 to ≈3.4 × 104 cells per ml. Eight 200-μl aliquots of each mixture were dispensed into randomly assigned wells in a 96-well plate, and optical density (OD) at 420-nm wavelength was measured periodically by using a VersaMax automated plate reader (Molecular Devices).
Replay Experiments.
In the first replay experiment, populations evolved under the same conditions as the LTEE. In the second and third replay experiments, cells were incubated on MC plates. We started the first replay experiment on the 3rd anniversary of Stephen Jay Gould's death; we ended it on the 66th anniversary of his birth. For further details, see SI Materials and Methods.
Confirmation of Cit+ Variants.
Each putative Cit+ variant was streaked on MC agar and Christensen's citrate agar to confirm its phenotype. Additional tests are described in the SI Materials and Methods.
Test for Frequency-Dependent Interaction.
We constructed mixtures of clones from generation 33,000 to test whether Cit− and Cit+ clones coexisted and, if so, whether they interacted in a frequency-dependent manner. For details, see SI Materials and Methods.
We thank N. Hajela for years of outstanding technical assistance and M. Adawe for help with media preparation; together they poured some 20,000 agar plates used in our experiments. We thank Francisco Ayala, Al Bennett, Rita Colwell, Bruce Levin, and Simon Levin for reviewing our paper before publication. This research has been supported in part by grants from the National Science Foundation (currently DEB-0515729) and the Defense Advanced Research Projects Agency "Fun Bio" Program (HR0011-05-1-0057).
- ↵*To whom correspondence should be addressed. E-mail: lenski{at}msu.edu
This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on April 25, 2006.
Author contributions: Z.D.B., C.Z.B., and R.E.L. designed research; Z.D.B. and C.Z.B. performed research; Z.D.B. and R.E.L. analyzed data; and Z.D.B. and R.E.L. wrote the paper.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0803151105/DCSupplemental.
- Received March 26, 2008.
- © 2008 by The National Academy of Sciences of the USA
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Madness of the Hatter
Roleplay Palace :: Writing :: Stories :: Fiction
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Madness of the Hatter
A sinister shadow sighed and slowly sunk into its tall, comfy looking chair. A fire of black flames was harbored in the fireplace, making the shadows dance along with the dark light the flames provided. It breathed slow and deep, the sounds of the air entering and escaping its lungs was eerie and old-sounding. Tick, tock, tick, tock went the grandfather clock, the only sound that could be heard clearly other than the slow, silent breathing of the creature that sat. As the ticking and tocking continued, the foot steps of a certain person grew louder and louder. The step's taken were soft and almost silent but they could still be heard.
"Tick, tock, tick, tock goes the grandfather clock. Tock, tick, tock, tick goes my walking stick. My dear old enemy, how is it you're still not as friendly? Oh how I wish you would try and not be an unsocial sinister fly." sounded a voice. It wasn't friendly nor was it hostile. It was simply filled with madness and rhyme. A pale silhouette, dimly lit by the blackened flames, appeared next to the tall chair. A walking stick, or a cane in other words, was in its hand.
"You've come to visit me? In such a dark sinister place?" asked a voice. But the voice was hard to define simply by a gender. Two voices sounded from the vocal cords of this creature; one of a male and a female. They echoed each other and both were filled with almost a certain weariness and old, brittle sounding. The silhouette spoke once more, "This is your home my enemy, the place where you make it homey. I have come for a reason you know, for I am never without one my dear foe. I am leaving this world you're changing and I assure you I know what I'm doing."
After a few moments of silence, the creature spoke, "So you've noticed already? I know you're the maddest one of all here but to be able to sense the change in only a matter of months? I was certainly wise to give you your role. But, I must ask; Why do you intend to die and leave your role to another?" After a moment or two or three, a chilling but certainly mad laughter erupted from the pale silhouette.
"A question from you, from the one who knows all too? I will keep my rhymes away and I will tell you what I have to say. I am not fit for my role anymore, I have lasted longer than I was suppose to. Give my role to my sister for she is far more ready that you could fathom in that pigheaded skull of your's." it bellowed. After the silhouette had finished what it said, it turned to leave. As quick as the light, a hand grabbed the arm of this figure. The fire grew brighter, turning into a light grey. The figure looked down, its oddly colored eyes meeting the dark red ones of the hands owner.
"What are you planning Hatter? You know you cannot go against me."
The Hatter grinned his Mad Hatter grin and said, "Why nothing at all, why so suddenly appalled? I have done nothing wrong, no nothing bad in so long. I'm simply waiting my dear foe, for when Alice comes back and when you take your fall. You Who Casts the Roles cannot avoid the prophecy, no this maddened world will not let you rein with such simplicity. So play the game of destiny and have your eyes see this worlds reality. I may not go against you no, but there is one who can do so. I leave you my hat and my stick, tock, tick, tock, tick. Tick, tock, tick, tock oh look at the time! I must go."
The walking stick and the hat of the Hatter were left behind on a long table ready for tea time. The Mad Hatter had left with his rhymes and riddles, laughing all the while until his laughter was heard no more. The Caster of the Roles sat in its chair, thinking over the Hatter's riddle. A frustrated sigh was let out before it stood. The flames blackened once more and the sounds that stayed were the pacing footsteps of the One Who Casts and the sounds of the grandfather clock.
Tick, tock. Tick, tock.
"You found my weakness."
"It's small knives!"
Karou Heiwajima
- Unknown Poet
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I thought I would begin my very first blog entry by sharing the bare bones of my vulnerability in its present state. On July 30, 2016, my beautiful son Ivan Roberto died in an automobile accident. My lovely boy was funny, handsome, an athlete and oh so clever. He had been my most trusted companion, someone who I could always count on, since the moment I gave birth to him at the young age of eighteen. I have always been a "problem solver". Once I am presented with a challenge I immediately go into "I can handle this" mode and move forward. The loss of my son has shown me, that I am not as resourceful as I once thought myself to be. Some things cannot be "fixed". There is no "handling" here, there isn't even any "healing" –there is only "dealing".
As I realize that I am in the very early stages of this immense grief I thought it would help myself and others, to share this journey with you as it presently exists. Everything changes and I expect that this (and I) will continue to change as well. In the United States there is this current rush to line up and "get back to work". Punch in, punch out, eat dinner, go to soccer practice, plan mini-family vacation, jump back "in line" and repeat. I have presently been de-railed. My train car has jumped off the line and I am observing the other trains moving forward and backwards and my train is too heavy to place back on the train line. I am an observer now.
When my son was born I was conscious and I watched as they pulled him out of my womb and he took his first breath of life and I looked at him and said adoringly "Hey, remember me?" referring to the nine months we had shared together while he grew within my belly. Then I had to face the ultimate nightmare. I watched as they lowered my son's body in his white rose covered casket six feet into the ground, deep into the Earth where they would burry my baby and where the person I once was had died with him, and would be buried as well.
Here I am. Suddenly dead and an observer of a strange new place. I can see where I have wasted time. Where I gave too much attention to the trivial things such as "relationship issues", "work issues", "financial" etc.. I can review my previous life and suffer over all of my regrets. If only I could have a "do-over", alas, do-overs" are not possible. I have no choice, I cannot "heal" from this, I can only learn to deal with living in this new world without my son. A world which is foreign to me and to which I find it difficult to belong in the same manner I had belonged before. Those of us grieving for our loved ones know this all too well. It's difficult to watch everyone engage in life as we had known it when the world as "we" knew it -has come to an end.
I have enrolled myself in a self-help group for parents whom have lost their children along with my ex-husband and I have also enrolled our fourteen year-old daughter in a self help group for kids her age. I feel we are all shattered pieces now and every single offering of support which helps us to adapt in our new world and new state of existence -must be embraced.
Not even a week after my son passed away I reflected upon our death. I observed it in the following manner on August 4, 2016:
"This message is for all those suffering and mourning for the loss of my young son Ivan Roberto -and for all those who never knew my baby but because you love me, you are mourning for my loss. Lift your hearts up! The Japanese have a custom called Kintsukuroi. Within this custom, when beautiful fragile objects are broken instead of discarding the object as useless, they are repaired with gold increasing the objects value. "Not only is there no attempt to hide the damage, but the repair is literally illuminated, a kind of physical expression of the spirit." My incredible friend Ron Hudson reminded me of this and added that at this moment when I am completely shattered that I have actually been bathed entirely in gold. This is so true. Although it must be said that right now.. Underneath this gold …I am deeply feeling the fresh edges of my new shattered and broken reality and I definitely do not yet feel "golden". What I feel is like I was buried with my son yesterday. But know this truth, I also feel like a NEWBORN today. My heart is filling me with strength so I can prepare to learn to live all over again, even shattered underneath. One of my son's wishes for himself was to be a better version of himself. I will fulfill his wish and be a better version of myself. I want all of you dear hearts with cracks in your lives to fill them with gold, to be stronger and more beautiful than ever …To be reborn with me -to be golden."
The end of the world happen for me on July 30, 2016, when my loving and sweet twenty-two year old son, Ivan Roberto suddenly died. I have now found myself navigating within a foreign planet. I am gathering my strength with each day that passes and learning how to breathe again; to walk, talk and live in this new reality without him. Wherever my boy is now, I know he would want me to do my best. What now? Do my best.
***Getting Help when one needs it is one of the Highest forms of Bravery. If you have experienced the loss of a loved one: a child, a parent or sibling.. ANYONE you are not alone. Below are some resources which may bring you comfort***


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Tia Carmen's Turkey Picadillo
Enjoy the holidays with good taste and health and be sure to find this recipe in the Healthy Latin Eating cookbook, available on amazon.com.
This amazing Turkey Picadillo recipe is inspired by Chef Angelo's Tia Carmen. It's bold flavors set the tone of Classic Latin American cuisine and ensure that you will not miss out on any heavy lifting in the kitchen.
Holiday entertaining is upon us and with Christmas and New Years coming up, try this easy party recipe sure to win over anyone with lean protein and tropical flavors.
Share the time with family and friends, not your oven! Buen provecho!
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