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BLyS Sequence Analysis

I’ve been playing with some sequence analysis and phylogentic tree construction programs recently because I would like to introduce these sorts of data analysis into my biology classes. As a sample protein, I decided to use BLyS / BAFF, a protein important in regulating B Cell numbers. I’ve always wondered about the origin of this kind of molecule, since working on it in grad school, and this seemed like a decent way to get some ideas about where it might come from.

The first thing I did was go to the NIH’s National Library of Medicine website: http://www.ncbi.nlm.nih.gov

It’s easy to search for any protein / gene / whole genome you are interested in examining. Knowing that BLyS is vital in humans and mice, I chose to start with the human sequence. I retrieved it as the following:

>gi|20196464|dbj|BAB90856.1| BLyS [Homo sapiens]
MDDSTEREQSRLTSCLKKREEMKLKECVSILPRKESPSVRSSKDGKLLAATLLLALLSCCLTVVSFYQVA
ALQGDLASLRAELQGHHAEKLPAGAGAPKAGLEEAPAVTAGLKIFEPPAPGEGNSSQNSRNKRAV

The easiest tool to find similar proteins in other animals is the Basic Local Alignment Search Tool for proteins, or BLASTp. Just using default settings, I pasted the sequence in the search field and hit go. (note, I actually just used the accession number, not the whole sequence)

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This retrieved tons of proteins with similar sequences from the vast database of sequence information, from which I chose several model species. One thing I wanted to do was to include several primates as a sort of internal calibration (assuming that they would all have very similar sequences compared to more distantly related species). I also wanted to get a few animals’ sequences who are quite distantly related to humans (frog and ground tit fir that bill)

Once I had a list, I put them all into a single text file and then used that in a second program. This time, I decided that the best ‘multiple alignment tool’ would be CLUSTALX. It’s been around for a while and can create data in a number of different forms. Besides, it’s free and versions are available for both mac and PC.

Again, for starters, I just accepted the default parameters and did a quick alignment:

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Obviously, there’s something odd about the canid familiars (dog) sequence, but before I did anything about that, I just wanted to see what a phylogenetic tree looked like. This is another thing that Clustal does well, it will export your sequence alignment as tree data in a number of formats, then I could plug that data into one final program. This last is a web based program that I access through a french site (but you can probably find it in a number of places). The program is called DRAWGRAM. It accepts alignment data and outputs a graphical tree representation of the alignment.

This is an important logical step… What I’m doing is asking for a family tree of sorts to be displayed that represents the relationship of the sequences I provided. We might want to assume that this also tells us how related the organisms that have these proteins are – and that’s not wrong, but it’s also not thorough as we’re only using ONE protein to make that assumption.

Here’s my first tree:

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Note how isolated Canis is on this representation.

Finally, I went back and truncated the Canis sequence to a place where I suspect the protein actually starts – my sequence from the NCBI gave me a string of Amino Acids at the front of the protein that I think are probably not there, but just got added by some computer algorithm without proper human oversight.

Once I did that Canis (by the way, I remained the sequence ‘DOG’ so I was sure it was the new one) fell in line with a sequence more similar to that seen in cats (felis):

ImageThat’s it for now. Although I expect that I will dig a little deeper with more animals to see if I can come closer to an ‘original BLyS’.

 References:

  1. Dereeper A., Audic S., Claverie J.M., Blanc G. BLAST-EXPLORER helps you building datasets for phylogenetic analysis. BMC Evol Biol. 2010 Jan 12;10:8. (PubMed)
  2. Dereeper A.*, Guignon V.*, Blanc G., Audic S., Buffet S., Chevenet F., Dufayard J.F., Guindon S., Lefort V., Lescot M., Claverie J.M., Gascuel O. Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res. 2008 Jul 1;36(Web Server issue):W465-9. Epub 2008 Apr 19. (PubMed) *: joint first authors
  3. Felsenstein J. PHYLIP – Phylogeny Inference Package (Version 3.2). 1989, Cladistics 5: 164-166
  4. Larkin,M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J., Higgins, D.G. (2007) Clustal W and Clustal X version 2.0. Bioinformatics, 23:2947-2948.
  5. Thompson,J.D., Gibson,T.J., Plewniak,F., Jeanmougin,F. and Higgins,D.G. (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 25:4876-4882.
 
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Posted by on March 7, 2014 in Uncategorized

 

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Evolution Animated

I just stumbled upon this cartoon for the first time today and I’m totally blown away. Sure, there are some things that could be explained better. There are a couple of moments when the illustrations could be a bit more accurate. But, overall, it’s a very good summary of the basic elements of evolution and pretty funny. (I wish I had made this!)

Have a watch and enjoy.

also, check out Kurzgesagt’s other animations on the Big Bang Theory, et al.

 
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Posted by on March 6, 2014 in Uncategorized

 

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Photosynthesis: Turning CO2 into O2 – or maybe not.

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It’s so simple, right?

“The evolution of photosynthesis remade the Archaean Earth. Before photosynthesis, the air and oceans were anoxic. Now the air is a biological construction, a fifth of which is free molecular oxygen”  – Bendall et al. 2008cIt’s easy to mistakenly think that photosynthesis turns CO2 into O2, people have been doing it for years. In fact, you’d even be remiss not to initially think that it’s the case – it is, after all, a simple conclusion to make and William of Ockham tells us to always start with the simplest ideas.

How could we do this experiment now?

We could  use radiolabeled Oxygen in our CO2 and then look for that same radioactive O2 being produced as a waste from the plant. But if that experiment were done, we’d quickly see that this wasn’t the case. As we will see below, this experiment was eventually what was done and instead of labeled CO2 being produced, the leaves of the plant becoming radio labeled, while only ‘cold’ CO2 was being released. Vexing!

One complication in addressing this idea comes from the very notion of air as being something to begin with. So, what is air? – and what happens (to air) during photosynthesis?

The Dutch scientist and physician, Jan Baptista van Helmont (1579-1644), did some early experiments to understand the nature of photosynthesis. His experiment was to determine where the mass of the plant came from. He suspected that it would be from the soil it was growing in, and did a very simple experiment that refuted this hypothesis. He reasoned that if the mass of the plant came from the soil, then it was a simple conversion that he could observe happening over time as soil was depleted resulting in an equal growth in mass of the plant. His experiment used a potted willow tree planted in 200 lbs of soil. In five years, his 5 lb sprig grew to 169 lbs, using only 2 oz. of soil.

Clearly the mass was coming from somewhere else. Knowing that he watered his tree regularly, he speculated that this was the source of the tree’s growing mass.

Helmont’s experiment did nothing to answer the question directly, but it does introduce a new player into the mix: Water… H2O. There’s Oxygen in water too – another possibility?

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What could possibly have killed this mouse?

In 1771 Joseph Priestley came onto the scene with experiments examining the nature of air as something more than just “nothing.” He noticed that a flame tainted the air with a kind of pollutant that was not amenable to animal life. He called this pollutant, phlogiston. Phlogiston could be produced by burning a candle in a closed container until the candle put itself out. Then, any animal (he used a mouse), that was put in this phlogistated air would quickly die. Yet a sprig of mint could counter this effect and somehow clean up the phlogistated air.

What do we know now?

1. Air is not just ‘nothing.’

2. Air quality (composition) is affected by certain biologic and abiologic processes.

a. Candle flames pollute the air with something toxic to animals (at least mice)

b. A mint sprig is sufficient to neutralize or eliminate this pollutant

Another Dutchman, Ingenhousz determined that de-phlogistation by plants occurs only in the light and required he green parts of plants to accomplish this.

(Much of the above material can be found in the excellent History of Research Page)

How to observe these gasses more easily? Perhaps under water, where gas will appear as bubbles.

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A simple experimental setup to measure photosynthesis

“When a sprig [of Elodea] is placed upside down in a dilute solution of NaHCO3 (which serves as a source of CO2) and illuminated with a flood lamp, oxygen bubbles are soon given off from the cut portion of the stem. ” -from a History of Photosynthesis. Using this device (pictured below) as a readout, F.F. Blackman measured gas production under various conditions by observing the production of bubbles under a number of conditions.

Data from such an experiment looks like this:

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The data

From these data, Blackman concluded that photosynthesis occurred in several stages, the first was a ‘light-limited’ stage that hastened with increasing light intensity, the second did not increase with increasing light intensity and required the work of enzymes (accounting for the effect of heat speeding up the reaction).

The Dutch scientist, van Niel  first suggested the idea of Oxygen gas coming from H2O based on his observations of purple sulfur bacteria converting H2S to S2 and assuming a parallel reaction was occurring in green plants.

CO2 + 2H2S → (CH2O) + H2O + 2S             (observed in purple sulfur bacteria)

CO2 + 2H2O → (CH2O) + H2O + O2             (predicted in green plants)

The final proof of this did not come until Ruben and Kamen were able to use an isotope of Oxygen to trace its route through photosynthesis.

Using algae, given ‘heavy’ oxygen in the form of either water or carbon dioxide, it was found that the isotope given in H2O was invariably that found in the resulting O2. Their experimental procedure is outlined in the diagram as two parallel experiments:

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% 18O FOUND IN
H2O CO2 O2
START 0.85 0.20
FINISH 0.85 0.61* 0.86
START 0.20 0.68
FINISH 0.20 0.57 0.20

So, what we should be saying is not that plants turn carbon dioxide into oxygen, but that plants turn carbon dioxide into sugar, which is precisely why van Helmont was confused by a 169 lb. tree growing from only 2 oz. of soil. He probably never would have believed that all that tree was actually built out of thin air.

 
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Posted by on September 28, 2013 in Uncategorized

 

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Magic Bullets

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from SAFC global

I just saw that the most recent issue of Science is highlighting a favorite topic of mine, antibody-mediated therapy and B cell biology. I’ve done work in both of these (related) fields in the past and remain an advocate of antibody-tageting of therapies using drug conjugates (e.g. trastuzumab–DM1) or initiating cell-specific effects simply by binding (e.g. anti-CD20). 

In the early 20th century, Paul Ehrlich coined the phrase “magische Kugel,” to describe antibodies as ‘magical’ proteins which could unerringly home in on targets to do a variety of things. Today, we can paint tumors with antibody conjugated with fluorescent dyes, deliver toxic chemicals to cells we wish to eliminate or simply activate / deactivate cells through targeting of receptor proteins.

I’m eager to get my hands on this issue and see what’s new (if anything) in the field and what products are currently in the pipeline of various biotech companies. 

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(I’m suddenly struck anew with the misery of not having access to Nature and other journals I’ve always had handy. I’m so glad I at least still have Science! )

 

 
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Posted by on September 15, 2013 in Uncategorized

 

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Coding Challenge

Recently, there have been a couple new revelations about number theory published in Science Within the article was a pair of theories about prime numbers that I had never heard before, one of which was:

Goldbach’s conjecture, [which] makes two assertions: that every even number greater than 2 is the sum of two primes, and that every odd number greater than 5 is the sum of three primes.

I thought it would be fun to start with the first part of this problem and write a program to accept user input in the form of an even integer > 2  and then look for the two primes whose sum is equal to the user provided:

Goldbach_partitions_of_the_even_integers_from_4_to_28_300pxprime1 + prime2 = user input

where prime1 and prime2 may be any prime number (even the same number twice)

I could easily see this escaping the processing power of my machine if the numbers get high, but I think it shouldn’t be too hard to at least write a code that could look for them and demonstrate whether this worked with known input.

Are you up for a quick challenge?

zombie locker

Learn Fractions with Zombies

If so, submit your documented answer here as a comment. Feel free to use any language you would like (I just did it in C++, but I’m eager to see better answers than my own). My favorite submissions will win a free copy of  my iBook, In Parts, Tales of Fractional Zombies, which you can enjoy yourself or regift to a youngster in your life who wants a fun way to learn the concept of fractions.

You can use these links as resources to help check your work:

prime numbers       prime checker

If you are new to coding and are looking for a coding environment to work in, check out this posting for help setting up a C++ coding environment using Xcode (on your mac)

 
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Posted by on June 19, 2013 in Codecademy, Coding

 

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Release of ‘The Curse of Sisyphus’

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The Curse of Sisyphus

The Curse of Sisyphus has been released and is available on the iTunes iBookstore. To celebrate the release, this, and its companion volume, The Thirteenth Labor of Heracles are both free until Sunday.

Zeus is not one to be trifled with. And Sisyphus has been a thorn in his side, defying him at every turn, yet escaping every punishment with uncanny cunning. But this time, the mortal has gone too far and Zeus has a special punishment befitting Sisyphus’ persistence.

The Curse of Sisyphus is the tale unlike others you may have heard about him before. Here you can find out exactly how Sisyphus defied Zeus yet again – and learn about the physics of motion, gravitation and orbit at the same time.

 
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Posted by on June 13, 2013 in Uncategorized

 

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Almost forgot

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Mercury, the closest planet to the sun has temperatures ranging from 400 degrees C on the surface to -170 degrees C in some permanently shadowed craters.

I was just about to go to sleep when I remembered that my biology class is having a quiz tomorrow and I should post a hint to an extra credit question. Rather than post it here, I’ll refer you to the Nature.com site that has extraordinary new data about a newly identified water reservoir in our solar system.

Follow this link to Nature’s news story about the finding.

 
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Posted by on December 4, 2012 in Uncategorized

 

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Aside

ImageScientific method as a lens to view the world                     

Science has a problem in telling its stories to the world. The problem stems from the way that science is done and the way its discoveries are published in academic journals not known for their mass appeal. In science, seeing something happen once or hearing about an occurrence might lead one to get in the lab and ask a question, but it is never itself acceptable as an answer. Instead, multiple repetitions of an experimental question are asked until consistent results are found under controlled conditions. Compare this to watching the news or reading the paper and you’ll find a drastic difference. Mass media loves the anecdote of ‘one family’s story’ or ‘what happened to my kid.’ Stories appeal to our natural tendency to relate to people and to incorporate the underlying morals or lessons into our broader worldview. He-said-she-said arguments are presented as fair and balanced even when balance is unjustified. With this in mind, consider this story of how the birth of the scientific method changed the world we live in.

The events of the late 15th and early 16th centuries ushered in a new age. Not just in the discovery of new land to be conquered and populated by western civilizations, but also in the way westerners saw the world and their place in it. Suddenly there was half a globe of terra incognita. As Columbus revolutionized our geography, Copernicus and Galileo revolutionized astronomy. But what was good for cartographers and astronomers wasn’t so good for the royalty and the church, who had been comfortably enjoying in the status quo.

At that time, the vast majority of people were ruled by the very few who held their power by divine right. What they said was both fact and law and any who differed in opinion did so at their own peril as autocracy seldom views challenge or change as good.

But cracks were beginning to show.

Between the discovery of the west and the mounting evidence for heliocentricity, came the greatest schism the Christian church has ever seen, hammered home when Luther nailed his 95 theses to the church door in Wittenberg, Germany 1517. Within a century a new world was discovered, the earth was shifted from the center of the universe to a body orbiting the sun and even God’s own voice on earth was being challenged due to a poor financial decision granting the sale of indulgences. How unfortunate for those in power that the printing press, which Johannes Gutenburg had introduced a half century earlier, made it so easy for this news to circulate around the globe amongst a newly literate population.

By the middle 17th century, Bacon and Descartes were formalizing the rules of logic and, more specifically, of scientific inquiry. Their texts, Novum Organon and Discourse on Method display a new desire for evidence-based reasoning rather than simply accepting facts ad hominem – even in cases where the hominemis the Pope himself. Bacon wrote, “The logic now in use serves rather to fix and give stability to the errors which have their foundation in commonly received notions than to help the search after truth. So it does more harm than good.” That is, too much effort is being spent defending what we already think is true rather than just following what the data tells us. This is exemplified by Ptolemy’s

How Ptolemy saw the solar system

complex system of interlocking circles and the complex movements they require in order to explain the wanderings of the planets in the night sky. An excellent demonstration of this backwards logic can be found at http://people.highline.edu/iglozman/classes/astronotes/retrograde.htm

With the upset in worldview brought by the renaissance and its new rigor for asking scientific questions along with a few intervening centuries, one would think that we would be more discerning in our beliefs today. We should be open to new ideas that challenge accepted dogma, and be in possession of tools to discriminate between unfounded speculation and well-supported theories.

Or, perhaps not.

In many ways, we are just as easily swayed by ad hominem arguments, faulty logic and satisfaction with the status quo as we were seven hundred years ago.  But it’s not entirely our fault. We’re not built to think critically, rather, we make quick judgments based on what we see in front of us. The ability to make split second decisions were likely required to save our skin in the time of Hobbes’ “state of nature.” While labored, methodical reasoning would reason us right into the lion’s mouth. The difference today is that we are vastly more educated than our forbearers, possibly smarter -if rising IQ scores can be taken at face value- and, frankly, we do have the time to practice methodical reasoning. Rarely do we need to make life saving fight or flight decisions in modern life.

Science has taught us that the universe may not be as we see it. In fact, our senses are fooled all the time. Sticks don’t bend when we put them in water, a continuous tone may appear to change pitch as the source approaches or recedes from the observer and quantum mechanics tell us that all the matter of the world is nothing like what it appears to be. Our senses tell a variety of lies to us, but nature does reveal her laws with careful study.

Understanding scientific method is teaching oneself to remain impartial to results of experiments, to ensure that the tests applied are rigorously designed to disprove  – rather than support – your hypothesis, and to rely on the power of statistics to interpret your results rather than being swayed by anecdote or a desire to see a predetermined outcome. None of this comes easily. As I said above, we are a storytelling people, stories are the lenses through which we view the world. It’s a lot easier and exciting to believe an anecdote than it is to understand the truth. But it’s often worth the effort and sometimes there might be an interesting story behind the science too.

An old essay I wrote about the scientific method

 
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Posted by on October 20, 2012 in Education

 

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Nature’s hidden beauty – A tangent from Intro Bio

Photosynthesis is a way that nature observes the first law of thermodynamics.

As we all learn in school, the sun is the primary source of energy on Earth, but only a fraction of Earth’s residents can tap into that energy directly. The rest of us, the heterotrophs (from hetero- other and troph – food), get our energy indirectly. We either eat the plants (or other organisms) that produce their own food, or we eat the things that somewhere down the line got their energy from eating autotrophs (from auto- self).

But, because the first law of thermodynamics states that energy cannot be created or destroyed, but only converted from one form to another, these autotrophs could not make their food from nothing. Instead, they converted (solar) energy from the sun into chemical energy via photosynthesis.

Solar energy, which comes to Earth as photons, has characteristics of both particles and waves (as it turns out everything does). These waves have energy that is inversely proportional to the wavelength of the light- shorter wavelengths transfer more energy than longer ones. I like to think of it this way: Shorter wavelengths mean more waves per unit time. If you were one the beach watching waves come in to shore, if more waves crash on the beach in an hour on Saturday than on Sunday, then more energy was transferred per hour from the ocean waves to the shore on Saturday.

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Absorption spectrum of pigments

The visible light we can see only a small slice of the broader electromagnetic spectrum. Because we see only the light that bounces of things, if those things absorb some of that light (such as plants that use the light for photosynthesis), then we see only what they reflect back because it is not absorbed. This explains precisely why most leaves appear green – all but the green light is absorbed by pigment molecules that are collecting energy in the chloroplasts.

We can see this clearly by looking at an absorption spectrum of several pigments found in leaves.

What’s really interesting, is the beauty of flowers. These parts of the plant are not photosynthetic*, but they also contain pigment molecules. Why?

Of course we know this. Flowers are the reproductive organs of plants, and they often require assistance from insects or other animals for pollination. The way they attract pollinators is by giving a reward (nectar) and providing visual cues about where that reward can be found (the colorful flower).

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Visual spectrum comparison

But, it turns out that bees (a common pollinator) don’t see the same visual spectrum as we humans do. Instead, their spectrum is shifted slightly in the ultraviolet direction.

Naturally, this would have consequences. If bees can see UV light, it would be reasonable to expect that some flowers use pigments that make them visible at UV wavelengths. In fact, this is exactly what we see – well, what we would see if we could see UV. Here’s a representative flower shown as we see it and as a bee may see it – with a UV colored landing area right where the pollen and nectar are found.

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Natural Light / UV Light

*At least I think they aren’t. If anyone can provide an example of flower petals that photosynthesize, that would be greatly appreciated.

 
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Posted by on September 28, 2012 in Uncategorized

 

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The nature of truth

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The Valley of the Shadow of Death

There was an interesting podcast by RadioLab this week concerning the nature of truth that I wanted to comment on. There were a number of stories in the broadcast, as always. One on yellow rain is getting the lion’s share of attention on the RadioLab site for the treatment of one of the guests that many listeners objected to. However, that is not the one I would like to focus on. In fact, I really only wanted to mention the podcast because I thought it was a good introduction to a topic that I find troubles a lot of people.

First, the podcast. I would point you towards the short, “In the valley of the shadow of doubt,” about one of the earliest photographs taken during wartime. In fact, the episode is about two photographs by the same person, Errol Morris, who was documenting the Crimean War in 1855. The two photographs depict the same scene, titled ‘In the Valley of the Shadow of Death’ that depict a road in the Ukraine. In one, the road is littered with cannonballs. In the other, there are no cannonballs on the road, but there are many off on the side of the road in ditches an on the hills.

The question that the photographs bring up is, ‘Which one was taken first?’ That is, did the photographer come upon a road littered with cannonballs that were removed – or did he come across a road surrounded by cannonballs that he moved in order to catch a more interesting shot?

Of course, we can never know.

There are reasons that can lead us to believe one thing or another (personally, I think one argument is stronger) but there is no way to know absolutely one way or the other. This is the real question that the episode brings up, “Can we ever know truth?” This is a very basic question in science. Most scientists agree (I am presuming) that we can never know anything with certainty. We can only rule out unlikely answers and give support to one theory or another.

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Wielder of Occam’s Razor

We can blame Descartes for starting this with his Discourses on the Method published in 1637. He started the trouble by giving us the scientific method, a method for uncovering the way the world worked. In his pursuit of this, he also realized that we cannot really know anything. He admitted only one thing that we are sure of. Cogito ergo Sum. But from this modest beginning, he also built up a structure and assured us that we have to assume that we can trust in at least logic, and that, until there was reason to believe otherwise, we may as well proceed as if the world we see around us does exist – evidence that he read his William of Occam (1288-1348).

Natural Philosophers and scientists have been fairly comfortable with this state of affairs for years. Assume that the theory with the most data supporting it is true up until the point that new data demands a change in thinking. At this point, we are instructed to drop the old idea and embrace the new one until it inevitably is displaced.

But these words mean different things to different readers. Some may read this as, “See, they admit it, they know nothing. And even worse, the are certain that their ideas will be proven wrong sometime in the future.” Others think, “Yes, of course. How else could one perceive the world?” And they’re both right – in a manner of speaking. It is assumed that much of what we know will change over time. But we also have the security of knowing that our understanding of the world is getting better all the time and it is unlikely that with new ideas we will entirely abandon out old ways of thinking. Rather, we expect to tweak this ideas.

And this would all be fine. But there is another school of thought that comes mostly from the journalists. That is the idea that every position / point of view is equally valid. There are a lot of questions that come to mind where I do think opposing ideas have equal value. These are political questions mostly. However, when journalists come to interview scientists about some finding or idea, two (or more) sides often don’t have the same weight of evidence behind them.

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…hungry……

If I were to ask you where teeth go after they fall out and are placed under a pillow, you might say, “The parents take them and give the children money.” You might say, “the tooth fairy comes and leaves the money in exchange for the teeth.” I tell my son that the tooth fairy needs teeth because she eats them and couldn’t survive without nourishment.

Not all of these hypotheses are equally likely. I have to admit that I’ve never seen the tooth fairy, but someone must have left a camera out to get this picture…

Back to RadioLab. So, what’s true? Does the weight of evidence make something true? Does it make it more likely to be true? Does evidence mean nothing?

On a deep level, perhaps we never know anything. But I can also say this: data is nature’s voice and sometimes it pays to listen.

 
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Posted by on September 26, 2012 in Uncategorized

 

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