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CRISPR: Accelerating the pace of molecular biology

CRISPR stands for Clusters of Regularly Interspaced Short Palindromic Repeats. Dr. Jennifer Doudna was one of the first researchers to see these short palindromic repeats in bacteria and archaea where she speculated that they were being used as a form of molecular immune system to protect these organisms from viruses.

Even bacteria get sick, so having a protection against invading viruses is a matter of life and death to a cell. Recall that viruses are essentially genetic material that will reproduce itself again and again after it hijacks a cell. Viruses may have protein coats or membranes to protect them outside of the cell, but inside, they are little more than DNA. If this DNA can be damaged or destroyed, then the virus is rendered harmless.

Screen Shot 2015-07-27 at 9.54.10 PMTo the right is a clip from Dr. Doudna’s video illustrating the repeated elements (in black) flanking a variety of ‘other DNA’. This ‘other DNA’ is what the cell will use to identify  foreign DNA – presumably from retaining the genomic material from an earlier exposure either in the lifetime of the single cell or its parents.

So, how does it actually work?
Two videos do an excellent job of explaining how CRISPR works. A short, simple video from MIT gives a non-technical explanation (a good place to start).

MIT’s McGovern Institute

Jennifer Doudna explains the system in greater detail…

Basically, the natural system uses two RNA molecules to target specific DNA sequences in the genome and recruit a protein that acts as an endonuclease to cleave this target:

crRNA – a ‘targeting’ molecule
tracrRNA – an adaptor RNA that recruits CAS9 to the bound crRNA
CAS9 – an endonuclease enzyme that will bind and cleave DNA once recruited by the RNAs

Doudna’s lab improved the system by combining the two RNA molecules into a single RNA that still effectively recruits CAS9 but is easier for researchers to manipulate in the lab. This last element is essential because manipulating this RNA sequence gives researchers the power to target any DNA sequence in the cell.

As stated above, the system was originally identified in prokaryotic organisms where it appears to allow targeting of the viruses that attack them. CRISPR uses ‘stored’ DNA as the targeting RNA and then brings in CAS9. CAS9 binds to the targeted DNA and cleaves it resulting in one of two possibly outcomes. 1) the virus is destroyed and is no longer a problem, 2) the virus is cut, but then repairs itself – hopefully in a way that introduced fatal mutations.

images
How might this translate into clinical medicine?
The possibilities are endless, however a few low-hanging fruit present themselves immediately. Among these are therapies for sickle cell anemia (and a host of other blood disorders). Because sickle cell anemia is caused by a single base pair mutation, it is conceivable that hematopoietic (i.e. blood) stem cells can be isolated, the faulty gene repaired, and then re-introduce the corrected stem cell back into the body (possibly after the faulty stem cells have been ablated).

The newly altered and re-introduced stem cells now do the rest of the work for you by finding their place in the body where they reside while continually producing cells with the desired genetic changes.

The key is that these RNA molecules are quite simple to make exactly and in pure form (i.e. they can be manufactured chemically rather than needing cells to do the job for us and then we have to clean up all the extraneous contaminants). Most labs will design the molecules in-house and then order the constructed molecules from a ‘core lab’ that specializes in doing just that.

Jacob Corn, of UC Berkeley has compiled a simple protocol that anyone with a modicum of molecular biology training could follow. Find that protocol here.

 
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Posted by on July 27, 2015 in Uncategorized

 

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Cool guys don’t sulk, they brood.

CalvinHobbesCool1
A rose by any other name…
My wife says that I tend to sulk when I feel mistreated.
Fortunately, I was able to correct her: Cool guys don’t sulk, they brood.

You know… because sulking is a bad thing that spoiled kids do when things don’t go their way or they don’t get what they want. And brooding is just being withdrawn from a world that doesn’t understand you (and it might even look a little like being bored, because the world bores you when you’re cool)

I was listening to the Stuff You Should Know podcast today about Road Rage, the phenomenon that hit the streets of LA and other big cities in the late eighties and into the nineties. At the time, road rage was making waves because of a spate of freeway shootings on highways in and around LA.

Today we call it aggressive driving and pretty much expect it from everyone (minus the shootings).

from the stuff you should know podcast:
http://www.stuffyoushouldknow.com/podcasts/road-rage-grrrr/

What I especially enjoyed was the suggestion that the best remedy to instances of rage was quiet contemplation (which was jokingly referred toin the 70s offered as the best solution for dealing with anger.

It made me wonder, what is the best response to anger and frustration? Catharsis? or meditation? Do we do ourselves harm by ‘burying it all down inside?’ or does letting it out simply condition us to being openly angry?

Dr. Arthur Janov, in Why You Get Sick – How You Get Well,  was a proponent of releasing anger so that it doesn’t eat you416WnwcbCfL._SX339_BO1,204,203,200_ up inside. He suggested that

“Repressed pain divides the self in two and each side wars with the other. One is the real self, loaded with needs and pain that are submerged; the other is the unreal self that attempts to deal with the outside world by trying to fulfill unmet needs with neurotic habits or behaviors such as obsessions or addictions. The split of the self is the essence of neurosis and neurosis can kill.”

The cure for this repression was to let it out in ‘Primal Scream Therapy’  that released the pain and allowed a person to become whole again.

[The Primal Therapy Website]  website
In 1998, a new trend in anger management philosophy entered the ring as Jill Littrell published an article, IS THE REEXPERIENCE OF PAINFUL EMOTION THERAPEUTIC (Clinical Psychology Review Volume 18, Issue 1, January 1998, Pages 71–102)?, suggesting that re-experiencing powerfully emotional memories can be therapeutic, but only in the controlled situation of psychotherapy designed to re-cast these experiences in a better light. That is, the value is not in catharsis, but in cultivating a less traumatic neural network that is called when remembering an experience.

To address the question, Bushman, Phillips and Baumeister write in a 2001 article in the Journal of Personality and Social Psychology (http://www-personal.umich.edu/~bbushman/bbp01.pdf) that their data supports the idea that aggressive people do engage in ‘anger-out’ expression in the hope that it will improve their mood (if they were told they were given a drug that would ‘freeze’ their mood, they tended to abandon their ‘let it out’ strategy.

imagesHowever, the same study found no evidence that this method actually worked to reduce anger.
The next year, followup work by Bushman, at Iowa State University looked at the effect of 1) Letting it out, 2) Holding it in (ruminating), or 3) Distracting oneself from the issue in reducing anger and aggression. It is interesting to note that both holding it in and letting it out resulted in poor outcomes, only distracting one’s self from the issue effectively reduced anger and aggression.

Perhaps ‘cool’ needs a new definition…

… not brooding or letting it out in a scream.  Maybe just wearing a sombrero will do.

calvin-hobbes-cool-2

 
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Posted by on July 26, 2015 in Uncategorized

 

Stephen King’s Carrie and the problem of genetics in an horror story

carrieIn the novel, Carrie, Stephen king attempts to explain telekinetic ability in terms of a real genetically inherited trait. OK, this is fiction, I have no problem with Carrie’s telekinetic ability … when would this story be without it after all?
Explaining this ability in terms of science was a mistake for two reasons. For one thing, it undermines the very idea of ‘supernatural’ that the reader has already bought into. This was exactly the problem that fans of Star Wars had with the prequel trilogy’s explanation of ‘The Force’ in terms of sub-cellular microorganisms. The second reason he shouldn’t have done this is because he didn’t understand it well himself.

Characters:
Carrie White – The protagonist, who possesses telekinetic (TK) ability
Margaret Brigham – Carrie’s mother
Ralph White – Carrie’s father

From Stephen King’s Carrie (please don’t sue me Mr. King)

It is now generally agreed that the TK phenomenon is a genetic-
recessive occurrence-but the opposite of a disease like hemophilia,
which becomes overt only in males. In that disease, once called “King’s
Evil,” the gene is recessive in the female and is carried harmlessly.
Male offspring, however, are “bleeders.” This disease is generated only
if an afflicted male marries a woman carrying the recessive gene. If the
offspring of such union is male, the result will be a hemophiliac son. If
the offspring is female, the result will he a daughter who is a carrier. It
should be emphasized that the hemophilia gene may be carried
recessively in the male as a part of his genetic make-up. But if he
marries a woman with the same outlaw gene, the result will be
hemophilia if the offspring is male.

In the case of royal families, where intermarriage was common, the
chance of the gene reproducing once it entered the family tree were
high-thus the name King’s Evil. Hemophilia also showed up in
significant quantities in Appalachia during the earlier part of this
century, and is commonly noticed in those cultures where incest and
the marriage of first cousins is common.

With the TK phenomenon, the male appears to be the carrier; the
TK gene may be recessive in the female, but dominates only in the
female. It appears that Ralph White carried the gene. Margaret
Brigham, by purest chance, also carried the outlaw gene sign, but we
may be fairly confident that it was recessive, as no information has ever
been found to indicate that she had telekinetic powers resembling her
daughter’s. Investigations are now being conducted into the life of
Margaret Brigham’s grandmother, Sadie Cochran-for, if the dominant/recessive
pattern obtains with TK as it does with hemophilia,
Mrs. Cochran may have been TK dominant.

If the issue of the White marriage had been male, the result would
have been another carrier. Chances that the mutation would have died
with him would have been excellent, as neither side of the Ralph
White-Margaret Brigham alliance had cousins of a comparable age for
the theoretical male ottspring to marry. And the chances of meeting and
marrying another woman with the TK gene at random would be small.
None of the teams working on the problem have yet isolated the gene.

Surely no one can doubt, in light of the Maine holocaust, that
isolating this gene must become one of medicine’s number-one
priorities. The hemophiliac, or H gene, produces male issue with a lack
of blood platelets. The telekinetic, or TK gene, produces female
Typhoid Marys capable of destroying almost at will….

Questions

Stephen King’s explanation of the genetics of hemophilia is not quite right.

1. How is hemophilia actually inherited? Explain in terms of dominant / recessive inheritance.
2. King suggests that hemophilia is inherited from two carrier parents. Is this correct? Describe, in genetic terms, how a boy can be born with disease.
3. Is it possible for a female child to inherit the disease?

Queen_Victoria

 
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Posted by on July 20, 2015 in Uncategorized

 

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Hot Cars in the SummerTime

I get ‘The Daily Upworthiest‘ each day in my inbox. This typically brings inspiring videos or ‘good’ news items as a way of not being inundated with the (Bad) News of the day. Today’s is important enough that it bears repeating.

In the Summer, cars can get extraordinarily hot in just a short period of time. Data by McLaren, Null and Quinn, published in the journal Pediatrics in 2005 demonstrated the rapidity in which car interiors heated up even in when the ambient temperature was not excessive. Using a dark-blue 2000 Honda Accord with medium-gray interior and without tinted windows as a test vehicle, they measured temperature rise over time. Even with ambient temperatures as low as 73 degrees F, the vehicle heated up to above 110 degrees F by 60 minutes.

F3.medium

Of course you would never leave your dog / child in the car for 60 minutes. Perhaps only 10 minutes tops, right? This still would result in temperatures above 90 degrees F, even on a cool day.

What about if I leave the window cracked? Admittedly, this helps, keeping the car up to 20 degrees cooler than if windows were up, but there was still an alarming spike in temperature.

Most importantly, these experiments were done under conditions without someone inside the car warming it up from the inside and filling it with carbon dioxide. That’s not an easy test to do seriously, however, here’s where The Upworthiest comes in… you can watch this video of adults being offered $100 for being willing to remain inside a car for just ten minutes.

A 107 degrees body temperature is lethal – something attained at a rate of about 37 cases a year since 1998. Most commonly, victims are less than 1 year old and were simply ‘forgotten’ in the car (slipped the caregiver’s mind while running an errand, or unintentionally left for hours.)

The message is clear, Never leave children (or animals) inside a closed vehicle, even when it is not particularly hot outside.

My favorite suggestion, comes from Morris Franco of Kars4Kids, who was quoted in the Upworthiest article saying,

Have a stuffed animal designated in the car seat always. When strapping in the child to the car seat, place the stuffed animal in the passenger seat. This will serve as a reminder to the driver upon reaching their destination that your child is in the backseat.

Have a good summer and stay out of the heat!

 
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Posted by on July 20, 2015 in Uncategorized

 

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Mozart and Harper Lee

I could really use some music to honor my wife on the anniversary of her death...

I could really use some music to honor my wife on the anniversary of her death…

In 1791 Mozart died while working on a beautiful piece of music, his Mass in D minor. I love much of Mozart’s work, but I think that this is probably my favorite (perhaps a tie with The Marriage of Figaro). Yet, there has always been discussion about how much Mozart, himself, completed and how much his friend and copyist (possibly student), Franz Sussmayr, wrote as he completed the manuscript for delivery to Count Franz von Wallsegg in 1792.

What is relevant here is that it does not really matter to me who wrote it. It’s attributed to Mozart, so I assume that he did the majority of the work in at least shaping it and providing hints as to how it would develop.

Similar accusations have been raised about Harper Lee’s authoring (or lack of authoring) To Kill a Mockingbird, and now Go Set a Watchman. Ms. Lee was good friends with another iconic writer, Truman Capote. The two were childhood friends, and she worked with him for some time as an research assistant for his opus, In Cold Blood.

To answer this, Maciej Eder and Jan Rybicki assembled a data analysis algorithm to analyze writing styles. With only a single novel, it is difficult to say much about the authorship (of Mockingbird), however, with the release of a second novel, a data analysis technique known as ‘cluster analysis’ becomes more meaningful. Using a number of analyses, the two data miners assert that Ms. Lee’s voice is her own, distinct from Capote’s. One of these analyses is presented below (taken from the Computational Stylistics Group website), examining most- frequent -word usages by Lee and a number of other Southern Authors.

Lee_and_others_consensus

We see that both of Lee’s books cluster together (as do other authors), and that her own style appears to more closely resemble authors that she professed were influential to her rather then that of her friend, Capote.

What is most important to me though is how I feel about the text. At this point I am nearing the end, but have not gone far enough that I can say definitively what my conclusions are. I admit that it took some time to get into the novel – the first chapter or so didn’t feel right to me – but most of the book has developed well in my opinion. I think what will make or break this book in terms of real importance to me is where things go with respect to the central question of race that it deals with.

Regardless of that conclusion, I have greatly enjoyed this book (as I did Mockingbird), for its ability to transport the reader into the mind and body of the protagonist, Scout. Taking us in a journey through time – twice! Once to Scout’s childhood, and again to her adulthood, still many years past now, just after World War II.

These books and Mozart’s Requiem Mass, they are what they are. And I intend to enjoy them by that standard.

The Requiem would be no less a masterpiece if it was written by Donald Duck. And Go Set a Watchman is what it is regardless of who wrote it or who wanted it published. The fact is, it’s out there and the whole world is devouring it this week. I say, discuss the politics of the book all you want, it’s all quite interesting too, but judge it on its own merits, irrespective of all these other questions.

That said… are you reading it? What do you think? I’ll probably be finished by the time anyone gets around to reading this, so answer as thoroughly as you like. Let’s consider this a SPOLIER ALERT for anything beyond this point – don’t read the comments if you have not finished the text. (OK, with all that lead up, I need some comments….)

 
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Posted by on July 17, 2015 in Uncategorized

 

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Casting a Long Shadow

thThere’s no pressure on Harper Lee.

No. She’s already published one of the world’s most beloved novels. Even people who haven’t read ‘To Kill a Mockingbird’ know her characters, Atticus Finch and his daughter, Scout.

And it’s only been half a century that her fans have waited for a second book, all the while singing the praises of Mockingbird:

It hardly seems like 50 years since I picked up this book late one rainy night…”   –     ,JLind555 Five Stars

“With its slow, warm and evocative opening chapters, Mockingbird starts off like a sultry summer day…” – Will Errickson , Five Stars

“…I am completely awed by this story.” –Dianna Setterfield, Five Stars

So, no pressure Ms. Lee. And, when your new book comes out tomorrow, I suggest that you turn off the TV, turn off the radio, and maybe read a good book.*

So far, all I know is the cover, and that looks like a good start.

watchman

*I realize this may sound callous after Ms. Lee’s stroke in 2007 that rendered her largely blind and deaf, but I certainly don’t mean to be.

 
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Posted by on July 13, 2015 in Uncategorized

 

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Pomplamoose – on making a living as 21st century artists

You have to wonder how anyone could think it is possible to make a living making music. Those at the top may as well have been born there – not because they didn’t have to work hard to get where they are, but because the number of artists who have ‘made it’ is so small that it may as well be limited to the winners of the PowerBall Lottery.

Pomplamoose is duo (Jack Conte and Nataly Dawn) making music and videos (I find it hard to imagine either one without the other) and doing the occasional tour. Last year (2014), Pomplamoose put together a 28 day tour around the US and then wrote up a summary of the accounting for the tour to illustrate how difficult it can be to make ends meet even for a relatively famous band (I admit that using the word ‘relatively’ could mean almost anything).

You can find Jack’s accounting of the tour and how their net loss of about $11,000 is just the price of doing business and building a fan base here. You can also check out Jack’s other project, Patreon, where normal people, like you and me, can be patrons of the arts.

 
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Posted by on June 25, 2015 in Uncategorized

 

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