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Do we have fundamental conflict in biology?
As saying "fundamental conflict", I mean something like two (or more) widely accepted results from different areas (or scales) can not exist logically together. In physics, there have been three conflicts1. I wonder if in biology, do we have such conflicts? And how biologists resolved that?
1  One of them is the conflict between Einstein's general relativism and quantum mechanics. String theory, if it is proved to be true, will reconcile this (read more). But it can also be like the controversy of the property of light. Some said it was particle, some said it was wave. Modern physics says it is both.

Several of them :P

Can you name some? I need a starting point to google

i think it is still not settled, whereas brain "computes" using mathematics, e.g. stores and solves equations, or process of vision is some stochastic MIMO black-box, which we represent as equations/math.models

But i think that googling should put you on right track. I haven't seen any relevant review, unless you consider latest Nature Methods article on p-value

This immediately came to mind, though it's not the example from different areas that you're looking for.

I think it is unlikely to have such (large scale/fundamental) conflicts in biology, because it is relatively easy to conduct experiments compared to physics. E.g. you wait for a proof of gravitational waves since when? Decades? The same with string theory, etc...

@canadianer Nice! :D We have exactly this kind of problems. I read many similar articles.

@aandreev this one turn out to be too technical to me...

Here is one of them : http://link.springer.com/article/10.1007%2Fs10539-008-9126-x
http://sharesend.com/xc55e117 - Hendry, Gonzalez - 2008 - Whither adaptation.pdf

Peto's Paradox is a great one. This addresses the disconnect in cancer incidence with cell number in an organism. It goes something like this:
Assume current oncogenesis theory to be correct in that mutation acquisition is the rate limiting step in creating a cancer, such that you need ~5 specific mutations in a single cell for that cell to be fully transformed into a cancerous cell. This then being the case, it is so rare that any cell can acquire all necessary mutations that in humans we don't see cancer incidence appreciably increase until the 5th decade of life.
Now, let's assume man has an average weight of 50kg and a blue whale has an average weight of 130000kg, with roughly equivalent lifespans. Assuming average cell weights are the same in both organisms, there are 2500X more cells in a blue whale than a human.
If linear mutation rate is the rate limiting step in cancer then increasing cell numbers is akin to increasing the number of times you flip a coin to try and get heads, which means there is a 2500X greater chance of getting all ~5 necessary mutations in a single blue whale cell than in a single human cell. If human cancer incidence starts to spike at 50 years, this means blue whale cancer incidence should spike after 7 days and blue whales should largely be eliminated by cancers within the first month of life.
Hopefully makes you question a bit what is causing cancer.
Make sense?

I never heard of that. It is interesting. However, it is the number of cell divisions that separate a given cell at the adult age and the zygote that matters and not the number of cells. Also, individuals that develop cancer rarely developed from a "clean" zygote. This greatly reduce your estimation as well. So is there still some kind of Peto's paradox or is it totally negligible?

@Remi.b -- More cells = more cell divisions required in the first place. Whale cells are not orders of magnitude larger than humans, despite being orders of magnitude larger as an organism. Since the vast majority of cells have some turnover, having more could also mean a hire rate of division even if the time it takes is higher. Peto's paradox looks fine (to me), but it does point out that DNA mutations do not always result in cancer. An extra stop-check prior to cell division seems to have a tremendous effect on cancers if you look at Naked Mole Rats. ;-)

Right, MCM is hitting a bit closer to the point. Remi.b when you say that the number of cells division is important, I agree, but once you have created an organism the cells do not stop dividing, they continue to divide and turnover for the rest of your life. And if you are suggesting by an "unclean" zygote, that it already carries something like an inactivating p53 mutation, this is a very special case resulting in serious problems for the organism because every cell in the body carries the mutation, and will oftentimes be embryonic lethal.

And I have to address the Naked mole rats because I think this is great evidence to the contrary of the mutation-centric cancer model. Naked Mole Rats have a higher density hyaluronin which may impede metastatsis, and hyperplasias. This is a great example of an evolved strategy to limit oncogenesis during potential reproductive years. And is doing nothing to change cellular mutation rate.

this one is something I'm looking for. Thanks for that.

Could not fit in a comment...
The question very much depend on what you mean by fundamental conflict (to the point that I am surprised to see nobody complaining that the question is primarily-opinion based). My first reaction is to think that there is no such fundamental conflict in biology just because biology is not really a "fundamental science". I am not sure what I mean by fundamental but I hope the comic will help understanding my point!

Of course, it is not true that biology is just applied chemistry (as I would expect the other relations to be wrong as well). Personally, I know nothing about chemistry and in my field of research (theoretical population genetics), we use math, statistics (which is just applied math) and algorithmic (which I guess is just applied math as well). Math is just applied logic by the way.
There are things we don't understand, but I don't think there are many fundamental conflict or paradox. There are many things that we have hypothesis (sometimes vague hypothesis) for it and have no idea how to test them.

i think question is about experiments that are contradictory, but haven't been refuted. Or contradicting hypotheses, both of which are supported by strong data and peer-reviewed publications. In terms of "fundamentalism", you are right, there is no "wave-or-particle"-type conflicts. Except on high cognitive level (see me comment to OP)

I always love this cartoon since I am in mathematics/engineering

@aandrev. I agree that there might have some cool fundamental looking stuff in neuroscience. It is a field that is in between algorithmic and neuroscience.

@dustin Yes, I like this cartoon. I'd appreciate to add philosophers even further to the right side. However, I apologize but engineering is by definition applied knowledge. So I would place engineering at a pretty low level of purity.

@Remi.b engineering is just applied physics if you think about it. The cartoon is also ordered in mathematical rigor so it would probably sit between chem and physics.

Have you read the explanation of it on explainedxkcd?

I think this is the most famous comic of xkcd. And I agree with @aandreev. So in your opinion, what is a "fundamental conflict"?

Well. I don't know but it is the OP's job to define the terms used in his/her question


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Where does the exudate comes from during inflammation?
During inflammation transudate and exudate is formed by vessels. I would like to make sure if it comes from arterioles, venules, or both, and the reason why.

exsudates almost exclusively come from the capillary compartment. If you think about the structure of the vessels, this appears logical.
The capillary compartment :

has vessels only several microns thick
has only the endothelial and basal lamina layers
spans an enormous surface, far greater than arterioles or venules

So when the fenestrations and tight junctions of the capillaries are impaired, serum directly passes into the extracellular medium.
A good example of that would be advanced anaphylactic shock. In this situation, the general inflammatory state causes a loss of endothelial integrity. Adrenaline given in such a state will have an inotropic and chronotropic effect on the heart, will cause vasoconstriction, but will also act to restore endothelial integrity. This last element is the main reason why allergic people carry an epinephrine syringe at all times.

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Where is pollenkitt secreted?
Where is the pollenkitt material secreted? I think the tapetum would be a good candidate, since it provides nutrition to the pollen grains.

This question may be closed, but I can't find the answer on Google Scholar. If it is closed as homework it seems to be difficult homework! I think this question deserves attention.

Thanks for paying attention towards my question. I really think it's tapetum but yeah I couldn't find any statement in support of this.

Pollen kit is secreted by tapetum

You'll find it in University Botany by Reddy shows up on first Google web search.

The correct name of the coating is "pollenkitt", not "pollen kit" (I edited your question accordingly).
From the book "Biology of pollen" (Shukla, Vijayaraghavan and Chaudhry, 1998):

The term pollenkitt is used for the oily, pigmented and sticky coating deposited on the pollen grain surface during the final stages of maturation. (emphasis mine)

That may explain why you and others struggled to find an answer in Google/Google Scholar.
Back to your question: there are two types of adhesive coats in pollen grains:

Pollenkitt
Tryphine

Pollenkit is the most common one and, as tryphine, it is secreted by the tapetum.
According to Pacini and Hesse (2005):

Two types of sticky pollen coat material exist in angiosperms, both produced by the anther tapetum. Pollenkitt is the most common adhesive material present around pollen grains of almost all angiosperms pollinated by animals, whereas tryphine seems to be restricted only to Brassicaceae. (emphasis mine)

Sources:

Pacini, E. and Hesse, M. (2005). Pollenkitt – its composition, forms and functions. Flora - Morphology, Distribution, Functional Ecology of Plants, 200(5), pp.399-415.
Shukla, A., Vijayaraghavan, M. and Chaudhry, B. (1998). Biology of pollen. New Dehli: APH Publ. Corp.

Yes it's Tapetum.Pollen Kit is made up of Hydrophobic pollen attractant and tryphine(A mixture of hydrophilic substances)

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Generate T2/T1 Phage Resistant E Coli
I would like to generate T2/T1 phage resistant Stbl3 E Coli to use in virus production. Is there a plasmid somebody has used that confers resistance, or is this done another way?

Two-step resistance by Escherichia coli B to bacteriophage T2 may not give you a specific protocol as to how to accomplish it, but there may be some useful info


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Advantages of gregarious vs solitary behaviour?
I'm trying to explain why birds (specifically wildfowl) are gregarious during the winter and solitary during the summer.
I think that in winter, birds are gregarious to reduce the risk of predation and to share foraging information. But gregariousness also brings with it competition, and because birds have offspring to feed in the summer, they trade-off increased predation/loss of information with reduced competition.
Does this sound correct?

what kind of effort did you put in answering yourself? have you asked google, e.g. "what are benefits of solitary in animals?"

You should add more information about your study species (living environments, migration, breeding behaviour  etc).

Question edited (hopefully improved)

sorry,. OP, it is still not good. Proper way to ask that might be something like that: "I wonder what is difference between X and Y. My hypothesis is Z, based on references A,B,C, and wild speculation D. Are there other theories?" Try to put it using as short sentences as possible and you will find help. This is not site for seeking assistance in editing writings

Another shot...

I will assume that you are talking about Mallards, although other wildfowl are likely to exhibit similar behaviors.
Mallards aren't actually solitary during the summer, instead they form breeding pairs.  They form groups (sords) in the winter to migrate and for protection while moulting.
https://sta.uwi.edu/fst/lifesciences/documents/Anas_platyrhynchos.pdf
http://thefarmatwalnutcreek.com/Ducks_Swans_Mallard-Duck.html


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What is the developmental basis of the placental sac?
The placental sac is the organ that provides nutrients to the embryo from the mother.
My question is that, since this obviously involves a lot of complexity, where does the sac originate from  (maternal / embryonic) ?
Also, what is the genetic basis of the above? do the genes intercommunicate between the embryo and the mother ?

I edited your question for it to be more in line with what you are after. I also updated the answer for the same.

We used to spend a few days studying the placenta in our embryology course. The short answer is, both.
Organ level embryogenesis:
The placenta connects the fetus to the uterine wall, and is the organ by means of which the nutritive, respiratory, and excretory functions of the fetus are carried on. It is composed of both fetal and maternal portions. The placenta can be traced right from the moment when the blastocyst is implanted into the uterine wall, though it takes a good portion of 12 weeks to develop.

After this adhesion, the invasion process starts, during which the cytotrophoblast cells from the trophoblast layer migrate deeper into the decidual layer. The formed vacuoles of the embryo become confluent to the uterus with the formation of the lacunar space 13 days after fertilization. The lacunar space will develop into the intervillous space for blood circulation a few weeks later. The placental progenitor stem cell is the cytotrophoblast cell. These cells are responsible for the cellular growth of the placenta, and they have the ability to proliferate and differentiate throughout gestation. The proliferating cytotrophoblast cells provide, after differentiation, the villous syncytiotrophoblasts as the outer cellular layer of the placenta.

I took the above paragraph from here (http://www.medscape.com/viewarticle/780615_2).You can read more about it here (https://embryology.med.unsw.edu.au/embryology/index.php?title=Placenta_Development.)
But for a serious scholarly study, you might be better off using textbooks.
Genetic basis of placental development:
As for the second part of your question, that is the genetic basis of development for a placenta, allow me to lift a paragraph from a book. You must remember that the embryo as it were, is not a distinct entity genetically, in that it contains half of the genes of the mother, and half of the genes from the father. And this is where biology becomes more complex, beautiful and truly mind boggling. It involves intricate interactions between evolution, organogenesis and developmental biology.

These results [in mice] led to an extraordinary conclusion. Paternal genes, inherited from the father, are responsible for making the placenta – maternal genes, inherited from the mother, are responsible for making the greater part of the embryo, especially its head and brain…
David Haig had begun to reinterpret the mammalian placenta, not as a maternal organ designed to give sustenance to the foetus, but more as a foetal organ designed to parasitise the maternal blood supply and brook no opposition in the process. He noted that the placenta literally bores its way into the mother’s vessels, forcing them to dilate, and then proceeds to produce hormones which raise the mother’s blood pressure and blood sugar. The mother responds by raising her insulin levels to combat this invasion, yet, if for some reason the foetal hormone is missing, the mother does not need to raise her insulin levels and a normal pregnancy ensues.
In other words, although mother and foetus have a common purpose, they argue fiercely about the details of how much of the mother’s resources the foetus may have - exactly as they will later during weaning. But the foetus is built partly with maternal genes, so it would not be surprising  if these genes found themselves with, as it were, a conflict of interest. The father’s genes in the foetus have no such worries. They do not have the mother’s interest at heart, except insofar as she provides a home for them.
The father’s genes do not trust the mother’s genes to make a sufficiently invasive placenta; so they do the job themselves…. Modern evolutionary theorists, led by David Haig, now think of the placenta as more like a parasitic takeover of the mother’s body by paternal genes in the foetus. The placenta tries, against maternal resistance, to control her blood-sugar levels and blood pressure to the benefit of the foetus.

Taken from: Matt Ridley, Genome: The Autobiography of a Species in 23 Chapters, Fourth Estate, 2000
For a more scientific explanation, I'd suggest reading the following papers.
Wang, Xu, et al. "Paternally expressed genes predominate in the placenta." Proceedings of the National Academy of Sciences 110.26 (2013): 10705-10710.
Hemberger, Myriam, and James C. Cross. "Genes governing placental development." Trends in Endocrinology & Metabolism 12.4 (2001): 162-168.

Does that mean that the DNA instructions as it were (sorry, not a biologist or scientist of any description) for the development of the sac come from both the mother and the embryo/fetus together? (When/if I get enough rep I'll up-vote your answer! Thank you :) )

Thanks for such a thoughtful and well written answer.  I take it from the Ridley excerpt that, effectively, the placenta is "organised" by the paternal genes of the embryo? I think that maybe my/our (am asking along with a group of pregnant English language teachers) biology defecit prevents us from understanding the finer points (that's probably  a massive understatement), so I hope that the last question doesn't sound silly. EDIT: have been advised to add that some are ex-biologists, of sorts.

@Bob If you are satisfied with my answer, please check the green box next to my answer to accept the same.

I certainly will! I'm a linguist and post a bit on SE EL&U. If it's ok with you, I'll wait a wee bit and see if any answers match your fantastic one? ... and then up-vote your answer!


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Difference between taking tissue of cancer from secondary place or primary place
Are there any differences when a surgeon takes tissue from secondary place (for example from metastasis) rather then from primary place (from an organ where cancer is) for morphological research?

I'm not sure I understand your question. Are you speaking strictly about research purposes, or diagnostics?

About research to determine if it's malignant tumor or not

Are you thinking about differences between a primary tumor and one which has already spreaded? I am not sure if I understand your question.

Yes @Chris i'm thinking about that.

I would disagree a bit with the previous answer. There are absolutely differences between the metastasis and the primary tumor. One result of this is that people very rarely die of primary tumors, but rather from the metastases of those tumors. The primary tumors will be quite heterogeneous and contain largely cells that have not undergone steps necessary for migration like epithelial to mesenchymal transition. After cells have metastasized they will be separate from the original primary tumor, and contain unique genomes. Because this is the more 'progressed' group of cancer cells, these are often the cells that need to be targeted. So, determination of the proper treatment strategy should be done on the metastasized cells.

I'm not talking about treatment. I'm talking about taking metastasis for research to determine property of cancer. I mean determine whether cancer is malignant or not.

Then you might be getting a little confused with terminology, if the cancer has metastasized it is malignant.

By definition, a cancer which has spread to another site via the circulatory or the lymphatic system (called metastasis) is malignant. If a surgeon takes it from a secondary site, it is malignant.
In the past, there were times that a cancer was so undifferentiated that to determine it's source was not possible. As science (particularly genetics) and technology continue to advance, tissue of origin is more easily determined.

Are there any differences when a surgeon takes tissue from secondary place rather then from primary place for morphological research?

Theoretically, it seems as it shouldn't, but it can happen at times, because, for example, a carcinoma may contain a mixed population of cancer cells, while the metastatic tumor may be of only one type.
For research purposes - which is different from diagnostics - it depends on the features most desired for research purposes.
In the past, there were times that a cancer was so undifferentiated that to determine it's source was not possible. As science (particularly genetics) and technology continue to advance, tissue of origin
NCI Dictionary of Cancer Terms
Multicenter Validation of a 1,550-Gene Expression Profile for Identification of Tumor Tissue of Origin

The surgeon said that it's not expedient to take a cell for research from pancreas ( there were Pancreatic Cancer ) and because it had already spread to a liver they took metastasis from a liver.
Is it the right method of approach ?

I would ask this on Biology.SE, where there are many researchers. Shall I migrate it there?

Yes please if possible.

@AlekoGharibashvili - Will do. Might take me a while because I'm a new mod. :)


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Identification of a plant (in a pot)
I have in my flat a plant which sometime make strange flowers. When the start it looks like plastic stars:

Then the flowers are opening after few days and looks like this:

The smell is a bit strange (I cannot say bad but definitively not good as other flowers). In general this plant does not need many watering and even I am in Switzerland, I guess it is living normally somewhere else.
For the size, mine is quite small (30 cm high but many wires) but my mother have a very long one along the wall (few meters).
Many picture with higher resolution are in my flikr gallery.
Anybody can tell me what is the name of this plant and where it lives normally?

http://en.wikipedia.org/wiki/Hoya

flowers looks like Asclepiadaceae

you could add image of its shoot and leaves.

It is a Hoya Carnosa Krinkle Kurl flower. It is native to Australia and East Asia. Hoya Carnosa links to wikipedia and Krinkle Kurl links to plant rescue in case you want to read more on it.


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During the process of correcting mutations via gene therapy, is the defective gene removed?
Just recently started learning about gene therapy, many websites explain that the corrected DNA can be added to the genome using a vector and all that. I just don't understand what happens to the other sequence of nitrogenous bases that codes for the incorrect protein.
Thanks in advance for any answers :)

To be specific: I am talking about adult, somatic gene therapy here, and germline gene therapy experiments is still a landmine when considering ethical reasons.
The defective gene codes for a defective protein, that usually plays a part in pathways. Since the protein is also defective, that pathway is also rendered defective because of this protein, and usually when pathways go defective, bad things happen.
There are three target approaches to gene therapy:

Gene silencing by Antisense Oligonucleotide Therapy
Genome Surgery by ZFN, Crispr/Cas9 etc..
Gene Replacement by viral vectors

The only method that corrects the defective gene here is the genome surgery method. By using Zinc Finger Nucleases, or Crispr/Cas9s, TALENS or other methods like that, the defective protein is corrected at the genome level, which automatically corrects the mRNA and protein, and everything works as it should.

Other methods like Antisense, just prevent the incorrect protein from forming. This is really useful if the defective protein is itself toxic to the body, and a replacement protein can be achieved by masking the defective region to produce a truncated but functional protein.

Gene replacement usually puts in a healthy copy of the gene in, that functions along with the defective copy, and this solves majority of the problems as the affected pathway is no longer rendered inactive, as the healthy protein takes over the defective pathway. The defective protein is still produced, but doesnt do anything (or rather, is rendered moot as the healthy protein takes over) and gets removed during regular maintenance of the cell.

Now depending on the type of disease the individual has, gene replacement may also take place along with gene knockout (depending on if the defective gene is toxic), but obviously many considerations have to be thought about before the combinatorial therapy can be used (toxicity of treatment itself being the primary concern, followed closely by efficacy).
Now obvious question with gene replacement, antisense or any other form of gene therapy is how we get the therapeutic agent to all cells, and yes that is a major challenge, and that's why gene therapy is expensive. And no, the therapy doesn't correct all the cells in your affected tissue, but it aims to correct majority of the cells, and that should restore function (atleast in myopathies like the muscular dystrophies)
As you can see here, in DMD, dystrophin gets muated due to a mutation We can either mask the mutation by Antisense therapy at the level of the RNA (which leads to a functional protein forming), put in an alternative form of dystrophin (mini or micro dystrophin) that takes over the function of dystrophin in majority (if we are so lucky) of the cells, and the overall phenotype of the individual improves. Recent advances in genome surgery are also being investigated for ameliorating disease symptoms. The reason I concentrated on DMD is that, gene therapy for DMD is my PhD. So apologies for the bias.

Very informative and helpful response, I really appreciate when people go above and beyond, and give well written answers such as this.


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Identification of a larva
Last week-end I was working in my parent's garden and when I dig a bit the ground, I found few larvas I never seen before. My father told me there are there every time when he's doing the garden springtime.
I took some pictures (I just put it on leaves for the picture to see better than on the ground).
One low-res:

And the gallery hi-resolution pictures
The location is in northern of Switzerland (Jura) at around 700 meters above the sea and the larvas were inside the ground in around 3-5 centimeters (1-2 inches) deep.

What you are seeing is a cocoon for a moth pupa.

Here is a link to a YouTube video of one hatching that is normal found in the soil.

Interesting, do you know how it look after?

@ruffp watch video link

Ok mine are a bit smaller (1-2 cm) but the video is impressive. But I don't know if there are still alive because it's not moving at all.


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Can we make a rough estimate of the number of generations since the origin of life?
According to what we've learnt :

we can approximate the number of generations since the first human pretty much like us (depending what / who we talk about).
we have an idea of the series of life forms we come from.

So is it possible to give it a very rough number ? Or it's not, because of some issues - but which ones ? (I only think about the many, short generations of the first forms, which moreover we don't really know about, as a limit to counting)
What if we stop counting at "unicellular" (or else) ?

Interesting question. Using the tree of life, we could get back as far as the last universal common ancestor (LUCA). I don't know if we can estimate generation times for early prokaryotes within an order of magnitude, though. Things get even harder before LUCA, because we don't know what life was like then. For example, how long did a generation last in the RNA world (if there was one)? Is the concept of a generation even meaningful in that world?

Perhaps we stick to an organism, that has been around a while, get its generation time, and calculate back from that. But as Corvus said, the more back you go, the more inaccurate you get.

shouldn't be too hard. see this http://biology.stackexchange.com/a/19569/8290

If you're willing to accept many orders of magnitude and define life as the Last Universal Common Ancestor.
For the rest of this answer, life begins 3.5 Gya with cyanobacterial mats and stromatolites and so on. Genetically the LUCA is dated to around this time, which matches the fossil record and everything's great. The LUCA can't have sprung from nothingness, so there may(will) have been (many)generations before the LUCA of things we would recognize as life, but I'm ignoring them. If they wanted to be considered they should have left descendants.
The fastest doubling times for modern bacteria are about 7-9 minutes but most are longer. E. coli takes about 20 minutes, and yeast takes hours. Given the 3.5 billion year clock, that gives an upper bound of about 1x10^14 generations. (Green sea turtles take 20-30 years to reach sexual maturity, or 1.1x10^9 generations). Something a bit representative is probably Pelagobacter ubique, which is vastly successful and takes about 29 hours to divide. Averaging out ice ages and so on and skipping the slowing of generations multicellularity probably implies(the Cambrian explosion is 82% of the way to the present day, being only 543 million years ago. We could account for it, but our guesses on generation time are way more inaccurate than any possible effect that could have) would estimate about 1x10^12 generations.

If they wanted to be considered they should have left descendants :-)

Averaging out ice ages and so on and skipping the slowing of generations Is it just hand-waving (I say that respectfully)? If not, I would be interested in math behind these statements

Nope, it's hand-waving. :( It would be nice to take into account but the largest source of error is the average generation time pre-cambrian explosion. Estimates of that need to be within 15% or so to outweigh the error introduced by assuming there has been one generation since the Cambrian explosion, and even tighter to justify accounting for a yearly spawn(starting in the devonian, maybe? I'd have to review the evolution of land animals) instead of just random growth. It's basically a significant figures issue.

This question has actually been covered (although not to the complete extent of evolutionary history) by Richard Dawkins in his book The Ancestor's Tale.
He gives the following estimates of LUCA time and generations:
Monkeys and apes: 40MY(3M)
Mammals: 180MY(120M)
Reptiles: 310MY(170M)
Ray finned fish: 440MY(195M)
Sharks, hagfish and lampreys: 530MY(240M)
Lancelets and all chordates: 560MY(270M)
However, he ceases to provide any generation estimates from here onwards, due to the error and general uncertainties being too large for a meaningful answer.

I like that this answer and my answer are both answers to the question but consider exclusively different creatures.


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Is it possible to express a mutant gene only in a specific tissue?
Imagine that someone tries to develop a knockout mouse for a gene, but this result in lethality for the homozygous.
Is it possible to express that mutant gene only in a specific tissue of interest to get a idea of which phenotype it produces? Does someone know an experiment which used this idea or something similar?

its possible, but it usually is unclear how to do it.

@shigeta can you expand?

I would like to comment on the language. You start by saying "express a mutant gene only in specific tissue". Then you mention specific mutation: KO. KO-gene is lack of gene expression. At the end you again say "express that mutant gene". I suppose you meant conditional KO.

While eliminating a gene is at least understood pretty well, tissue specific expression is difficult because the DNA sequences that encode the logic in animals as to when these genes are active are poorly understood.  Its been worked out only for a small number of cases I believe.

@shigeta You mean that you can gene-trap tissue markers, but we don't know how certain tissue is being form or what it consists of?

@aandreev  sorry i'm so swamped I can't do a proper job, but tissue specific promoters may only express a gene for a moment in development (and never again), or not be entirely specific for a given organ.  since we can't look at the DNA sequence and predict what the pattern of gene transcription will be, you would have to watch the rest of the organs over an entire lifetime to be sure (or several lifetimes).  a lot of work!

It is called conditional mutation.
You flox (put lox sites around) gene of interest and express Cre recombinase driven by tissue-of-interest-specific promoter.
Illustration from here:

Using chemically-activatable variant of Cre recombinase (cre-ER) you can create knock-out in some cells of tissue of interest, not every.
Addition: a bit weird but still useful method is to produce KO by means of CRISPR/Cas9 system. Tissue-specific expression of Cas9 protein together with gene-specific sgRNA will likely produce KO in mosaic fashion. One of positive sides is that such approach requires only one transgenic line, e.g. Tg(promoter:Cas9;sgRNA)
Addition: (via WYSIWYG's suggestion) you can express shRNA or other gene transcription-interfering product from promoter that only active in specific tissue/specific period of time. And beauty of genetic/bioengineering is that you can combine several methods for additional control, robustness, efficiency etc.
Taken from here:

Inducible and sell-specific gene KO with shRNA
For further reading:
http://cre.jax.org/introduction.html
http://link.springer.com/protocol/10.1385/1-59259-065-9:477#page-1
Update
I re-read OP question and, as commented to it, feel some confusion. So I would like to mention as well way to express mutated gene in specific tissue that, when expressed constitutively (all the time, everywhere), is lethal. Imagine some very strong oncogene. Or something like cell-toxic protein, e.g. chemically-activatable nitroreductase or light-activatable Killer-red. Or it might be mutated version of your favourite synaptic protein that, when expressed in all neurons, causes severe lethal phenotype.
What you do, is create transgenic animal with transgene-GFP fusion (nitroreductase, KillerRed, etc) that has floxed stop-codons in front, and cross that animal with transgenic with tissue-specific CreER (for example). What you get is animal that, when injected with tamoxifen, start to express mutated gene/toxic protein in tissue of interest. Then you are sure that only GFP-positive cells express that gene. That is how you "express that mutant gene only in a specific tissue of interest to get a idea of which phenotype it produces".

Additional info: For knockdowns, shRNA can be expressed in the desired cell type

Note that to get a knockout with Cre/LoxP system requires one further cross since F1 is heterozygous for the floxed gene. You need to cross your Cre +/0; target +/flox mice from F1 with the target flox/flox of F0 to generate Cre +/0; target flox/flox in F2.

yup, that is laborious


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Determining how much energy a plant takes in?
I asked a question on WorldBuilding.SE, "Are diamond berries possible?".
This led to asking a question on Chemistry.SE about the amount of energy required to burn a 1 ct. diamond (−6.527kJ).
The helpful commenter on WB.SE said that a plant would roughly require three times this amount of energy to essentially reverse the process through some biological means to create the diamond berry.  And that I would need to know the "wattage input" of various plants to compare with my hypothetical plant.
Is there a way to arrive at how much energy various plants take in?
Also any other related information from a biological perspective would be helpful.

Just a speculation for whether diamond berries could exist or not. This is not an answer for "what is the energy uptake rate of plants" because the actual question tends to be "whether a plant can gather enough energy to form diamonds". For energy uptake rate of plants please see MCM's answer.
Even though enthalpy of formation is same as heat required for destruction, burning a diamond is much easier than creating it (I presume it is easier to generate high temperature compared to high pressure). Natural diamond requires both high pressure and temperature to form. Synthetic diamonds are made by chemical vapour deposition; that also requires high temperature. Such high energies cannot be produced by any known organism (requires devices like arc furnace), and moreover at these temperatures the biological structure will disintegrate.
However there is one possibility of diamond berries; the berries would not be solid diamond though. A plant could be growing in an area where there are a lot of nanodiamonds (meteorite rocks) which could be bioaccumulated in the berries. Living organisms do accumulate nanoparticles [1,2]. There have been studies on biological application and effects of nanodiamonds too [3,4]. However these would not look lustrous like bigger diamonds. Xylem vessels in trees have a mean diameter of 30-40µm; it may be possible that the plant can take up bigger sized diamond particles. Density of diamond is ~3g/cm3 — three times that of water and it would require thrice the force required for water to get diamond to the destination.
For a spherical diamond of diameter 20µm:
Cross sectional Area = 314.15 µm2
Volume = 3351 µm3
Mass = 3×3351×10-12 ≅ 10-8 g
Gravitational force on the diamond = 9.8×10-8 ≅ 10-7 N
Pressure required to counteract gravity = 10-7/314.15×10-12 ≅ 3.18×102 Pa

However, I am not sure if that much of pressure can be generated within a vessel and whether small particles can be lifted like this.
There is another possibility that bigger diamonds are encapsulated by living tissue (not the fruits of course).

What I think would be a rough estimate (ignoring the physical/thermal requirements for diamond creation) TL;DR -- It's about 68W/m2 given the caveats below. Also:
Disclaimer: This is the best way I could think of to figure it out. It is a rough estimate only, and your level of desired precision will introduce more complicated calculations than the ones I've done. Not all of the calculations I performed are explicitly necessary, but were simply the best way for me to wrap my head around it at the time and may be incorrect!
Since plants are mostly glucose and water you could find the net glucose content of the plant (which will vary greatly -- but let's give a rough estimate of 90% as an average). So if you have a 10kg plant, ~1kg of the mass will be a mixture of cellulose, starch, and other stuff.
For the sake of ease, let's ignore the other stuff and focus on the cellulose and starch which we'll say makes up the 1kg. Both Cellulose and Starch are Glucose configurations, so what we have is 1kg of Glucose.
According to Wikipedia the net amount of energy a plant devotes to biomass construction/CO2 fixation is 3-6%, and the Gibbs Free Energy conversion of CO2 to Glucose is 114kcal. If I've done my math correctly that means a mole of CO2 to Glucose for a typical plant takes 2280kcal (or about what I burn in a day as a 6'4" man).
So, how many moles of Glucose is in our 1kg Glucose plant mass? The molecular mass of Glucose is 180.16g/mol. 1kg/180.16g/mol = 5.55 moles of Glucose. We don't need to know how many moles of CO2 we started with because we're not limited there; the atmosphere provides all the CO2 we could ask for. We just know that we have 5.55 moles of Glucose, and it took 12654kcal of energy to convert however many moles of CO2 into that many moles of Glucose.
That 12654kcal is also equivalent to 52944.34kJ of energy. Now, the solar constant is ~1.36kW/m2 ... or (1360J/s)/m2. So, how much area of your hypothetical plant is exposed to the Sun? A square-meter isn't an unreasonable amount for larger houseplants, so let's just work with that.
52944.34kJ / 1.36kW = 38929.66s = 648.82min = 10.8 hours' worth of Sun exposure to produce 1kg of plant mass. Some species of Bamboo can grow 91cm/day, but I couldn't find a source for density... but nearly 3-feet of bamboo could definitely weigh 1kg.
Sooooooo... Plants "take-in" 1.36kW/m2 when exposed to sunlight. How large the plant's area is will determine how much energy it takes in overall. How much energy a plant devotes to producing its mass is about 3-6%... or about 68 Joules every second (68 Watts using 5%) for a large plant with 1m2 exposed area.
Your 1ct diamond would take just over a minute and a half if 100% of the plant's mass-growing resources went into it.
Of course, that seems really fast. The important assumptions are that the plant is in direct sunlight, is not limited by other factors (Nitrogen is actually a major rate-of-growth limiter, as-is Phosphorous), halts all other growth-oriented metabolic activity to do so, and the metabolic processes for the conversion other carbon forms to Diamond was 100% efficient with the energy the plant demarcated for growth.
In reality about the only potentially "true" assumption from that list is that it's completely possible to have a plant with 1m2 of surface area exposed to sunlight. Everything else will be a fraction of what the working assumptions are.


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Synteny, genetics?
Could anyone explain the concept of Synteny relating to genetics? A picture would help. I tried read the wikipedia source along with another PDF
http://gep.wustl.edu/repository/course_materials_WU/annotation/About_Synteny_Analysis.pdf
And I feel it only somewhat helped. From what I gather synteny is about the order of genes, relative to their homologous genes? Or their location in general?

Same relative location i.e order in different chromosomes. Also have a look at homologous synteny blocks

Syntenic blocks contain the same genes of order between chromosomes of different species.

The figure above shows (left to right) syntenic block shared between human chromosome 17 and corresponding chromosomes in three other mammals (horse, pig and chimpanzee). And as expected, the more distinct the species (such as pig and horse) the more disarranged the order of genes are.
Ref: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3758187/

What do you mean by "the same genes of order"?   Did you mean something like "the same genes, in the same order,"?

yes same genes, same order.


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Why is my peripheral vision better for seeing in the dark?
I often walk around my house at night with the lights turned off and one night I noticed that looking straight at an object with very little light available made the object disappear from sight but if I directed my visual else where I could clearly see the object in my peripheral

"Peripheral vision is more light-sensitive than the central vision" written by AliceD here gives a great answer to your question.

Its down to the structure of your eyes. The eyes are made of rods and cones, which have differences in detecting light.
Now, compare the images below.

As you can see, the peripheral vision is predominately due to your rods, and your central vision is made of cones.

Measured density curves for the rods and cones on the retina show an enormous density of cones in the fovea centralis. To them is attributed both color vision and the highest visual acuity. Visual examination of small detail involves focusing light from that detail onto the fovea centralis. On the other hand, the rods are absent from the fovea. At a few degrees away from it their density rises to a high value and spreads over a large area of the retina. These rods are responsible for night vision, our most sensitive motion detection, and our peripheral vision.

(Taken from: http://hyperphysics.phy-astr.gsu.edu/hbase/vision/rodcone.html)
You can read about the photorecptors of the eyes here:
http://webvision.med.utah.edu/book/part-ii-anatomy-and-physiology-of-the-retina/photoreceptors/

This is not the complete story; this answer is more complete under linked question

Because peripheral vision uses the peripheral retina,which has mostly rods as photoreceptor,while central vision uses the foveola,a central part of the retina which mostly has cones as photoreceptors.
Rods are more sensitive to light. That is why they are more efficient in the dark.

all_licenses: Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/


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Can a relationship exist that is mutualistic/parasitic/etc. but not considered symbiotic?
Wikipedia definition of symbiosis: "Symbiosis (from Greek σύν "together" and βίωσις "living")[2] is close and often long-term interaction between two or more different biological species."
Wikipedia definition of mutualism: "Mutualism is the way two organisms of different species exist in a relationship in which each individual benefits from the activity of the other."
In intro biology class, I (and many other people, I'm sure) was taught that mutualism is a type of symbiosis. However, now that I think more about it, it seems like some relationships that are mutualistic don't appear to fit the definition of symbiotic. Some relationships are clearly mutualistic and symbiotic (humans and gut flora, for example) but other relationships (like bees pollinating flowers) are mutualistic but don't appear to fit the definition of "close and often long-term interaction". The same of course goes for parasitic, ammensalistic, etc. relationships as well.
Thoughts, anyone?

please, rephrase your question in more succinct forma than "Thoughts, anyone?"

TL;DR: mutualistic/parasitic relationships are members of symbiotic relationships class
So, first of all, there is a way to look it up.
Secondly, the very same article in wiki has subsection on parasitism. Reading it would be useful:

This is also known as antagonistic or antipathetic symbiosis.

Also, brief search through Google Scholar will show how these words are used in context. papers include such as:

Fungal symbiosis from mutualism to parasitism: who controls the outcome, host or invader?
Mutualism and parasitism: the yin and yang of plant symbioses
Mutualism or parasitism? The variable outcome of cleaning symbioses
Enhanced growth as a manifestation of parasitism and shell deposition in parasitized mollusks. Aspects of the biology of symbiosis.

Thanks for the answer. Now that I think more about it, it seems to me that all parasitisms do involve that close and long-term interaction, but I'm still not sold that all mutualisms fit the given definition of symbioses. What I'm asking about is whether an interaction that is mutually beneficial (like bees pollinating flowers) but doesn't actually involve "a close (physically) interaction" should really be considered a symbiosis. Perhaps that definition of symbiosis is incomplete? Or maybe the brief interaction between the bees and the flowers is enough for it to qualify as "close"?

what do you mean by "brief"? they won't live without each other, that is close enough for my taste. Again, these is how terms are used in specific field. Only reason to redefine them is discovery of a counter-example.

Brief as in the amount of time the bee is in physical contact with the flower is a short amount of time, compared to the lifespan of the bee and the flower, in contrast to gut flora, where the bacteria and the human are in physical contact for the entire duration of both organism's lives. It is, I suppose, not close enough for my taste, which is why I wanted to ask for some other opinions. I'll admit it's not the most exciting of questions.


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Is homosexuality an adaptation in humans?
Homo sapiens have menopause - as some other mammals (e.g. some Cetacea) - a phenomenon where older females live for decades after the end of their own reproduction.
Perhaps homosexuality, that looks to hold at 10% levels in any population, may be just the same sort of adaptation?

A quick scan of global and US data on wikipedia suggests the rate is closer to 5%.

To answer this question, we need to know the 4 values in the pay-off matrix, that is nb of babies of hetero sibling of a hetero, hetero sibling of homo, homo sibling of homo and homo sibling of hetero.

Also see: http://biology.stackexchange.com/questions/7669/how-can-homosexuality-evolve-despite-natural-selection

@kmm thanks for that link. Should we mark this question as a duplicate then? unless we edit this question to focus exclusively on kin selection ?

I would put between 5 and 10%, @user137. But homophobia makes lots of homosexuals deny themselves (lowering the numbers in most researches). And other biological phenomena occurs at the 10% threshold (left-handedness, black jaguars, left-turning shells, etc.)

Is there a reason you expect black jaguars and homosexuality to occur at the same rate?

@canadianer, as I said, many minorities in Nature occur at 10% of the population. And homosexuality is also around this number. See, for instance, http://en.wikipedia.org/wiki/Demographics_of_sexual_orientation#Brazil.

Barnard's wonderful text "Animal Behavior: Mechanism, Development, Function and Evolution" offers some enlightening summary to the discussion of homosexuality in a biological context!
Brief Summary to ch 12, page 606:

What we know so far:

homosexual behavior is recorded all throughout animal kingdom
there are evidence for genetic basis to homosexuality in various species including humans

Three interrelated schools of thoughts:

Homosexuality is maladaptive - external stress creates homosexuality

reduce resource stress on family unit, youngest child tend to be gay (E.M. Miller 2000)
*** mitochondrial DNA carries male killing genes, with male being a dead-end (Hurst 1991)

Homosexuality as an adaptive early phase of development - homosexual acts are just target practice

homosexuality is just males practicing sex on each other, most homosexual acts are performed by bisexuals with just as high fertility
  as straight males (Baker & Bellis 1995)

Homosexuality advantages in other aspects of reproduction - homosexuality may imply better parenting

homosexual genes induces feminine attributes in males such as sensitivity, empathy and kindness hereby increasing likelihood of long
  term pair bonding and paternal care (E.M. Miller 2000)

*** original text:

A completely different suggestion is that homosexuality may reflect
  the interests of selfish genetic elements (see 2.4.3.2) inherited
  maternally on mitochondrial DNA for which males are effectively a dead
  end. Some insects have been shown to carry 'male-killing' genes on
  their mitochondrial DNA, so perhaps homosexuality is a similar, if
  more benign, rout to sidelining males (Hurst 1991)

keep in mind these are ot mutually exclusive either, non-exclusive homosexuality can increase reproductive success, exclusive homosexuality is rare enough it could be a side effect of this beneficial effect. homosexuality is not a single behavior.

There is a study that found that female maternal relatives of homosexuals have higher fecundity than those of heterosexuals [1]. This suggests that an allele predisposing to homosexuality may propagate because it increases reproductive success in some situations, not necessarily in the homosexual individual.
However, many factors can influence a persons sexuality, such as birth order (in males each additional older brother increases the odds of homosexuality by approximately 33%) [2], so genetics only tells part of the story, and I found no comparable explanation for female homosexuality.
Here's an interesting article if you want further reading [3].

Camperio-Ciani et al, 2004. Proc Biol Sci. Evidence for maternally inherited factors favouring male homosexuality and promoting female fecundity
Cantor et al, 2002. Archives of Sexual Behaviour. How Many Gay Men Owe Their Sexual Orientation to Fraternal Birth Order?
IFL Science. Born this way? An evolutionary view of ‘gay genes’

Genetic view of adaptation:

Do note that this is looking at the view from the POV of a gene. Please read the comments below this answer, for a small discussion on the kin selection hypothesis. Do note that I do not study evolutionary psychology, or work on the level of the population.

This is a bit of a misconception, as nature really doesn't work like that. Nature works by allowing people to pass on genes, and then selecting those genes which are the best. Now the issue with homosexuality when viewing from an evolutionary angle is that, homosexuality by definition doesn't allow for genes to be passed on. I would also quote Richard Dawkins in this issue

“That’s a very tempting error,” Dawkins replied, “not with respect to
  homosexuality, but with respect to the idea that nature does
  population control. That’s not the way nature works.”
“It’s so tempting to think that things will be best for the species,
  so nature will do whatever it takes to keep the species going. But
  that’s just not the way evolution works.”
“Natural selection,” he continued, “works at the level of the
  individual gene. So in order for natural selection to favor population
  control, [it] would have to favor a gene or genes within an individual
  that limited that individual’s reproduction.”
“Homosexuality would do that, but of course, it wouldn’t actually be
  naturally selected because the individual [who] doesn’t have children
  doesn’t pass on the genes for not having children.”
“In order for natural selection to favor population control,” Dawkins
  said, “it would have to be the case that an individual who has too
  many children ends up rearing fewer” successfully.
He noted that this is exactly what ornithologist David Lack found when
  he studied avian clutch size — those birds that laid too many eggs
  were not able to care for all their hatchlings, whereas as those who
  laid too few were not selected for the obvious reason — they didn’t
  produce enough progeny.

Taken from: http://www.rawstory.com/2015/03/richard-dawkins-explains-homosexuality-didnt-evolve-as-natural-population-control/

The OP is referring to kin selection processes (or group level processes if you prefer). Selection does not only act at the level of the gene. Homosexuality might be beneficial if sibling of homosexuals have more babies. The allele would indeed increase in frequency. From Hamilton and Nash work, we can calculate the equilibrium frequency of homosexuality from the pay-off matrix.

@Remi.b Interesting you mention that. I was reading up on [Bobrow, David, and J. Michael Bailey. "Is male homosexuality maintained via kin selection?." Evolution and Human Behavior 22.5 (2001): 361-368.]  and [Rahman, Qazi, and Matthew S. Hull. "An empirical test of the kin selection hypothesis for male homosexuality." Archives of sexual behavior 34.4 (2005): 461-467. ] and they seem to find no support for this? Do note that I am not an evolutionary psychologist, so I am not an expert in this field and am not upto date on literature. Has supporting evidence been documented ?

It's also incorrect to assume that homosexuals never pass on their genes. There's nothing about being gay that makes you physically incapable of reproduction.

@user137 Yes, I agree that homosexuals can reproduce. But do note that homosexuals, can not reproduce, unless there is a partner of opposite sex involved (which goes against the scientific definition of homosexuality). What I mean is, if a homosexual chooses to reproduce by in vitro fertilisation, surrogacy or any of the other ways that currently exist, though legally a homosexual, there has to be a biological partner of the opposite sex involved. (I mean no disrespect to the LGBT community).

Yes, I'm thinking about kin selection. Rover Eye, Bobrow & Bailey found that "homosexual men were somewhat more estranged from family members, especially from fathers and oldest siblings". This seems a consequence of homophobia, not of homosexuality itself. I don't think there was homophobia in the environment where homosexuality evolved. Rahman & Hull used a very reduced sample (n = 120) in a country well known for its historical homophobia.

Also, we don't have to think of homosexuality and heterosexuality as two isolated concepts. They're more of two extremes in a continuum.

@Rodrigo, I presume you are referencing the Samoan study, where homophobia was absent? It does make sense in an obvious way doesn't it. The inclusive fitness offered by homosexuals, is largely dependant on acceptance of society, and inversely related to homophobia. And apologies for not being able to critique the papers properly. This is not my field, and I tend to work at the genetic level, not population level.

Ok, Rover Eye, no problem at all!


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Would it be possible to create a gel, holding viruses, that could alter the human genome when rubbed in?
I was wondering if it could be possible to engineer a virus, that when it comes into contact with human hair follicles, it could alter someones natural hair color.
Naturally blue hair anyone?
Doubtless there are loads of problems, but i was hoping they could be identified and potentially worked around.

You might take a look at Topical Gene Delivery on pubmed, but this also has papers that deliver to the eye, and any in vivo gene delivery is really hard right now. Also, blue pigment is not something you could easily make biologically. Blue birds use light interference to get their color.

So, i would need to somehow engineer biological microstructures, in order to reflect only a blue wavelength?                                                                          And when you speak of birds, you refer to plumage coloration, while i'm asking about hair specifically. There are some mammalian species that have blue or bluish hair. I'm wondering how to adapt that to a human.

Mamalian species with dye colored blue hair? I doubt that. This is probably some light effect.

Cats and rats can have a color that is classified as "blue", but it really looks more grey. Behold: http://messybeast.com/colour-charts.htm


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How is protein cavity centre related to binding?
I am confused and I have the following questions:
1. What are (in the context of article below) protein cavity centres?
2. How are they related to binding?
(Automated identification of protein-ligand interaction features using Inductive Logic Programming: a hexose binding case study, Santos et a. 2012)

I don't think this question should be closed. Yet you might want to add a bit more information and references.

no need to get hostile. There is a reason number of votes required for closing/putting on hold question. Regarding question: OP, do you have any reference to the phrase used? Isn't "cavity center" is just central point of given cavity?

As you can find under the table with cavity centers in pdf version of this article:

The table lists the protein’s PDB ID, the ligand considered and the specified cavity center. 22 ligands are similar to hexoses in shape and/or size. The cavity center is the centroid of the reported PDB atom numbers.

And a little later:

The binding-site center is computed as the
  hexose pyranose ring centroid for the positive examples,
  and as the ligand or empty pocket centroid for the negative
  ones. The hexose pyranose-ring atoms are located up to
  2.9
   ̊
  A away from the ring’s centroid. Since some atomic
  interactions can be important up to 7
   ̊
  A [22], we consider
  the binding-site as all protein atoms present within a 10
   ̊
  A radius sphere around the binding center. All other atoms
  are discarded.

So basically @aandreev is right. You can read about centroids here

I could not understand the head or tail of this, can you please tell me if there is any site which clearly explains these calculations?? I am not a biology or chemistry student, I am a computer science student, but my research field is bioinformatics... So i am having a lot of trouble trying to grasp what this thing means....

Could you be more specific, about your problems?

like where would I find information (calculation and explanations) of obtaining centroids of bonding sites of ligands, I want to know about these binding mechanisms and how these calculations are carried out..

https://en.wikipedia.org/wiki/Molecular_binding, https://en.wikipedia.org/wiki/Category:Geometric_centers

no no, I am probably not able to explain what I am having a problem with. Like what I want to know is that how did you obtain this fact "  The hexose pyranose-ring atoms are located up to 2.9 ̊ A away from the ring’s centroid. " . How did you obtain the the ring centroid, from which database and how did you calculate this, I know the general formula for centroid, how did you find it for the ligands

Well, if those guys didn't write it there, they probably thought of it as details - used standard formulas and atomic coordinates.

I did not understand what you meant by thought of it as details, how do one obtain these coordinates and calculate the cavity centres ?? They mentioned about a database, but could not find anything valuable, or probably I did not understand..

Atomic coordinate are in PDB files of proteins.

Sir , I could not find the atomic coordinates in the pdb files, can you tell me how to get the atomic coordinates?

@rishika https://www.cgl.ucsf.edu/chimera/docs/UsersGuide/tutorials/pdbintro.html


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Any evolutionary explanation for human blood groups?
What is the explanation of people having blood types from an evolutionary perspective?

I don't think blood group has any selective advantage (except for cases like duffy). IT seems like a neutral polymorphism that remains in the population.

Oh I didn't know about duffy. Thanks for sharing.

Researchers have puzzled over the selective explanation for the ABO polymorphism at least back to the 1950s. Multiple lines of more recent evidence, including the very old age of the ABO polymorphism and (perhaps) multiple convergent evolution events, suggest that the ABO polymorphism is maintained by balancing selection (E.g. Saitou and Yamamoto 1997, Segurel et al 2012). As best as I know, is no general consensus on what is driving balancing selection, though some researchers have argued that the selective agent is Plasmodium falciparum (e.g. Cserti and Dzik 2007).


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How can a heart of human work if one of its valve is not working?
Yesterday I went to a hospital and heard a doctor say that one of the heart valves of a patient is not working. However, the patient was still alive and was healthy; the patient could walk and talk too. I could not understand it because I have read that all valves of heart are extremely important. I have tried searching on google but all I could find was heart attack problems.

See the following articles on wikipedia to get your head around that topic: aortic insufficiency and aortic stenosis. You can also look for stenosis or insufficiency of the mitral, tricuspid, or pulmonary valves, all on wikipedia.

It is not possible that one of the valve is completely closed and the person is still alive without being in ICU. I think what the doctor was trying to communicate to the patient was that one of the valves is in the process of complete closure. There are three main types of diseases associated with heart valves :-
1- Blood flows backwards
2- Heart valve opening becomes narrow
3- upper condition may lead to complete closure
For details see the following link:-
http://www.nhlbi.nih.gov/health/health-topics/topics/hvd
http://en.wikipedia.org/wiki/Pulmonary_atresia
Hope you understand. :)

"Not working" can also mean that the valve cannot close, instead of being permanently closed.

@MarchHo Even if the valve cannot close properly, will there not be problem in blood supply. If what I am saying is right then patient should be in ICU. But as I said before patient was looking fine.

@NigHterz If the insufficiency or stenosis of the heart valve becomes severe, it will lead to death even with extensive care in the ICU. The only treatment is valve replacement, either by surgery or interventional cardiology. However, people do just fine with a minor defect. For example, a minor degree of mitral insufficiency can be found in almost 30% (depending on sources) of people, and they often never even learn about it in their lifetime. They live normally and practice sports.

@NigHterz 'But as I said before patient was looking fine', These problems often develop gradually and the impact gradually increases. The patient you saw may be fine as along as they are sitting, but they may be unable to climb a flight of stairs or walk more then a few dozen yards, without feeling short of breath.


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Finding Rna-Seq dataset for C. Elegans (Caenorhabditis elegans)
I want to find a RNA-Seq dataset for C. Elegans to use it in RNA-Skim software. For those who does not know RNA-Skim let me explain it a bit. It is a tool which is used to quantify RNA-Seq data using sig-mers(special kind of k-mer). Firstly RNA-Skim clusters protein coding genes and find substrings that are special to cluster of that gene. After this preperation stage, it takes an RNA-seq data as an input and tries to quantify transcripts in it. So, I need to find a dataset in fasta file format for this step. Since I'm pretty new in this area, is there anyone who could provide me information about how could I find it?
Complete information about RNA-skim can be found here : RNA_SKIM
EDIT
The type of a dataset I'm looking for is something like that: http://codepad.org/n3ioT3nj .This dataset is for mouse, I need to find such a dataset for C. elegans as I said before

Try any of these: www.wormbase.org, www.modencode.org, data.modencode.org, or ftp.modencode.org. Also, they won't be fasta files for NGS data they will either be fastq format or SAM/BAM format

Go to NCBI-SRA or GEO and search for RNAseq data. Set organism as C. elegans and optionally any other keywords that you are interested in. This search term will give you all RNAseq data in SRA from C. elegans: ("caenorhabditis elegans"[Organism]) AND "strategy rna seq"[Properties]

You will generally find files in fastq format which you can easily convert to fasta. There are many tools that do that; you can also do it with your own script. Following awk command will do the job:
awk 'NR%4==1{print ">"substr($0,2)} NR%4==2{print}' data.fastq > data.fasta
Apart from SRA you can also look at ENA (European Nucleotide Archive) and DDBJ

,thanks for information.As you said, I searched on the NCBI-GEO and when I typed C Elegans RNA-seq, I saw 65 results in this link : http://www.ncbi.nlm.nih.gov/gds/?term=C%20elegans%20rna-seq  , from them I clicked the first one however I could not locate the fasta or fastq file you mentioned earlier. Could you help me ? As it is my first time interacting with such databases I have some difficulties.

SRA is the right place for sequencing data. GEO will have all kinds of expression data. In SRA you just have to click on a particular run and find the experiment ID (Starts with SRX). Paste it in this page and download the data.

@stackunderflow In the GEO page when you click on one of the experiments you will get a SRA link at the end of the page. You can click on that (ftp link) and download your file. But these will be in .sra format which you have to convert to .fastq. .sra files are smaller and will download faster and you can use fastq-dump of sra-toolkit to convert sra to fastq. Or else paste the experiment ID in the second link that I mentioned in the previous comment.

thanks a lot, I started downloading a fasta file from the link you provided. I have one more question, As you know, I've tried to find a rna-seq data and I have already shared rna-seq data for the mouse specy in my question (http://codepad.org/n3ioT3nj ) Now, I tried to figure out what do these numbers in that file explain to me. I mean how could I understand expression level of something by looking that file. For example I could not understand nothing from the line 1:4807788-4897909W:ENSMUST00000150971:8:789:94:209:S/1. How could I learn it?

And also, mouse rna-seq data that I share link of it to you is just 160.2 kb but the file I'm currently downloading already exceed the 620 Mb. Do I download wrong thing ?

@stackunderflow ENSMUST00000150971 is an Ensembl transcript id. It is your fasta header and I am not sure how you got it like that. RNAseq fasta headers are not like that. They usually have information about the platform and run details. Is this fasta file generated after mapping? Then in that case the header denotes the alignment details i.e the transcript id, location -> 1:4807788-4897909 for genomic location i.e in chromosome 1 from x to y and then the location in the transcript. I am not sure why 4 numbers in the transcript location. S/1 and S/2 are for mate pairs.

this file was not generated by me. I got it from the following web page : https://github.com/zzj/RNASkim/blob/master/src/test_data/fa_reader_test.fasta.1 .I guess they put it as an example for mouse specy but they do not provide any information about this file. Unfortunatelly I could not run RNA-SKIM software for mouse species since it requires 48GB memory at least. So I decided to use C. elegans and in order to use it I have to generate such RNA-seq data for the C. elegans species. Since I have limited characters here, I summarized it here: http://codepad.org/NSIw0Oq2

in the first step, I made a typo, it is : they clusters genes according to sequence similarities*

do you have any idea ? Actually, If I know what are these 2 seperate two files, I think I'll figure out the rest. These are 2 seperate RNA-seq dataset or part of a single rna-seq data ? If the latter one correct, why they are seperated into 2 files. I could not understand.

Actually, I got it right know.  I think they use ,as you mentioned earlier, sra-toolkit to generate such 2 files. Since I'm still downloading sra file I have not seen the result of sra-toolkit yet, but I've read that fastq-dump executable of sra-toolkit produces 2 fastq files with "1" and "2" read suffices. These suffices are also consist in the name of files that comes with RNA-skim software

@stackunderflow Paired end sequencing data has 2 files and reads in one file will have the suffix "1" in the header while the other will have "2".

I have another question related with my main question. As you can see from the link http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE60731 I have found a RNA-seq data. In the sample sections, it is written that P7 Cerebellum DNase-seq Rep 1. There are also Rep2 and Rep3. What are these Rep extensions used for ? I could not find any information on the web. Could you please explain it?

@stackunderflow These are replicates of the samples. Sometimes same sample is loaded twice and you have two runs for the same sample. This is not the case here. Rep1 is one sample and Rep2 is another sample for the same experimental protocol. If you read the paper corresponding to the data you will understand. See the study summary (click more).

thanks for the information, according to your answer, there are two possible cases, in first case I'll have two runs for the same sample. Is there any relationship with number of runs that I have and type of reads (Paired-end or single)?  Furthermore how could I know whether these "rep"s are two runs for the sample sample or they are different samples ?

@stackunderflow two runs of the same sample will be under the same experiment SRX but the biological replicates will usually have a different experiment ID. These things are usually described in the summary

I think I have found 4 runs of the same sample in this link : http://www.ncbi.nlm.nih.gov/sra/SRX145660 ( 4 of them have same SRX ID as you said). But all of them have different sizes. I guess these 4 file are not identical right? If I make RNA-seq quantification experiment on these SRR files seperately, will I get same results from all of them ?

@stackunderflow they are same samples loaded multiple times. You can analyse all of them. The profile would be the same.

Okey, if they're same I do not need to do it, but I wonder why they have different file sizes while they are same samples ?

@stackunderflow because the loaded amount may be different.


text:
Mutations/deletions with CRISPR
I need to stop some protein from being active and searching for some universal way to do so. In mammalians.
With CRISPR it is possible to knock-out the entire gene. But it's a little complicate (need two gRNA for eg.). There are also another reasons, so I need to do this with only one gRNA.
So I am thinking about inserting some mutation.
My question: Is there way to make some mutation that will prevent creation of functional protein, with high efficiently - with one gRNA. And without need to know the active site of protein.
For example, frame-shift or mutation around start codon? Is it possible? If so, how can I do this? To where I need to send the gRNA?
Thanks.

First of all, If you talk about system, please specify if it is cell line or animal. Secondly, if you say "There are also other reasons", specify them too. Also, what do you mean by "without need to know the active site of protein"?

Well, synthetic CRISPR/Cas systems do have an option of having a single guide RNA (sgRNA)

You can do with a single gRNA. All that CRISPR-Cas, ZFN or TALEN systems do is to introduce a double strand break at a specific site. The DNA gets repaired via two mechanisms — non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ is error prone and it may introduce indels that can compromise with gene function (frameshifts etc). While HR can be used to integrate a transgene or repair a mutation, NHEJ can be used to create mutations.
I haven't really knocked out a gene using CRISPR-Cas but it is good to target the initial part of the ORF. There will be many other factors too that will determine what site should be chosen for targeting and an important factor would be the uniqueness of the target sequence. See this site for guidelines on selecting a good target sequence.
You would have to do rigorous screening nonetheless; the advantage of insertional knock out is that screening can much be easier (for e.g. by inserting GFP or puromycin-resistance gene).
You can have a look at this paper and the supplemental information; they have targeted 18080 genes with a CRISPR-Cas library.

Huge disadvantage  of insertion is very poor successful rate. Less then 5%. So I still looking for the answer. Where in the gene to cut, to make protein not working. About ORF, I wrote about start codon, but need more information and sources. Where exactly. Thanks.

And about the uniqueness, off topic question, is it possible to make a gRNA that will target a lot of different places in genome? Didn't heard about that. Nonspecific gRNA?

@VGranin the insertion rate you refer to is inserting a specific sequence, a non-specific indel mutation to cause a frameshift like WYSIWYG is talking about can have success rates upwards of 70%. And the only way you could target a lot of different sites with a single gRNA is if all the sites had a sequence in common, gRNAs are specific to a given sequence.

@VGranin As I said you can use a single gRNA to make the cut. Definitely ahead of the start codon but not too far off (if frameshift on non-sense mutation happens then you  will just have a little bit of the original sequence). So you have to optimize between closeness from start codon and other factors such as unique site and others mentioned in the link that I have given in the answer.

@Luigi. Thank you. But he mention a GFP insertion... Anyway, what CAS9 do is just making break. A non-specific insertion it is not something that we do, it's the cell way to repair. What I need, is a place to target gRNA to make a break that will make protein non working.

@Luigi Are you sure about the 70%; it depends a lot of factors including cell type and cell cycle stage amongst others.

@VGranin I mentioned that you can do an insertional mutagenesis too which, although will require two gRNAs, make the screening much easier (you can FACS sort the knockouts).

@WYSIWYG for a straight ds knockout, we usually see between 40% and 70% successful depending on the target

@Luigi perhaps we should discuss this on chat but which cells are these?? And how do you screen? PCR? Screening is the most irritating thing

@ WYSIWYG, thank you. So near the  start codon. But is there some paper that did this in different situations and proteins - and show some numbers of success e.t.c? Cause I can't find.. Thanks.

We use puromycin resistance to positively select for transfected cells, after this selection (with transfection efficiency now 100%) is when we see 40%-70% success with PCR screening (which is definitely a pain). @VGranin this paper will tell you everything you ever need to know about performing CRISPR knockouts

@VGranin this paper too. They have done a genome scale knockout for 18080 genes (not at the same time of course)


text:
D/L configuration for amino acids
Why would this be "L-cysteine"? This is taken from the answer key for my biochem final.

From what I understand if the -NH3(+) is on the left then the alpha-amino acid is in the L-configuration.
Am I wrong, or is my prof wrong?

We can look at the list of amino acids on wikipedia for a start. And we can look at this L-alanine:

What makes your image confusing is that it's a Fischer Projection, and I hate those because you have to remember what way the stereochemistry goes. In Fischer Projections, vertical lines face away from you, while horizontal lines face towards you. So if we convert the L-alanine I provided to a Fischer Projection, first we have to rotate the alpha carbon to put the methyl side chain in line with the rest of the carbon backbone. Now we look at the alpha carbon with the NH2 and H looking at us to create the Fischer projection. Here, the amine is on the left, not the right. You drew the amine on the right, which would give you the opposite orientation. If your professor had the amine on the right, I think he was wrong.

Additionally, the D vs L nomenclature is confusing. I prefer the S vs R nomenclature because that's what I was taught for molecules in general. Most amino acids fall into the S configuration, but cysteine is an R, because the sulfur atom has higher priority. However, cysteine is still an L, and the amine would still appear on the left in the Fischer Projection. This is why biologists stick to D and L for amino acids.


text:
Bayes theorem for mutations
MEN 2A is a dominant inherited disease caused by a mutation in the RET proto-oncogene.
The probability of being sick when you have the mutation of the RET proto-oncogene varies with age and is assumed to be 40% at 40 years of age.
With autosomal dominant inherited diseases, this formula can be used, where p indicated penetration and D is the sick allele:
P(II inherits D given that II is healthy) = $\frac{1-p}{2-p}$
The uncle of the son III1 in the tree has the disease so he assumes it comes from I1 or I2. The father of the son II2 is 40 years old with no symptoms.
What are the odds of the father having the mutation?
What are the odds of the son having the mutation knowing the % in the first question?

I just used the formula above as so for the first question: $$\frac{1-0.4}{2-0.4} = 37.5\%$$
I'm having problems with the second though and I assume to use Bayes formula.
I've tried as so:
$$P(\text{son mut given dad mut}) = \frac{P(\text{Dad mut given son mut}) \cdot P(\text{son mut)}}{P(\text{Dad mut})}$$
I know the probability of the dad having the mutation is 37,5% and the probability of the dad having the mutation given that his son has the mutation is 100%. However I can't seem to know what to put in the probability of the son having the mutation? Any help appreciated, thanks! Sorry for a long question.

We don't know the probability of being sick given that you are homozygous for the mutant allele so we can I guess just consider this to be negligible, so we'll assume that the kid is heterozygote. The allele either come form the dad or the mother with equal probability. the odds are therefore $\frac{0.5}{0.5}=1$. If we were to know that the father (and let's assume the father only) is carrying the mutant allele (let's assume in one copy only), then the probability of the kid to get this allele is 0.5. The odds are $\frac{0.5}{0.5}=1$ again. Did I missunderstand the problem?

I don't understand your calculations and I don't understand why you're trying to use Bayes formula. I don't know the $\frac{1-p}{2-p}$ formula and I don't understand what it is supposed to calculate. It seems to me that you're overthinking a simple problem.
We don't have all the information and need to make a bunch of assumptions but if I understand the question correctly then..
Probability that the father carries the mutant allele
I neglect the probability that a new mutation occurred and that the son is the first carrier of the mutant in the lineage. I assume that we have a probability of 0 of being sick if we don't carry the disease. Because MEN is a pretty bad disease, the autosomal dominant alelle causing the disease is probably kept at pretty low frequency. From wiki, I read that MEN 2A has a frequency of 1/40000. This means that the probability of being homozygous mutant is $\left(\frac{1}{40000}\right)^2 ≈ 10^{-9}$. I think we can safely assume that the sick kid is heterozygous. Given that the kid is heterozygous, the mutant allele comes either from the mother or from the father with equal probability. So the probability it comes from the father is $0.5$ and the odds are $\frac{p}{1-p}=\frac{0.5}{1-0.5}=\frac{0.5}{0.5}=1$
Knowing that the father is heterozygous, what is the probability that a given kids receives this mutant
By the law of segregation, the probability is 0.5 and the odds are 1.


text:
Do any birds have established "poke a predator's eyes out" behavior?
Is there a pattern/tactics of behavior in any birds species that consists of attacking a predator's eyes with its beak?
I'm looking for established tactics, not 1 or 2 anecdotal examples.

Not that it matters, but I arrived at the question when trying to answer http://scifi.stackexchange.com/questions/87740/how-came-fawkes-know-how-to-disable-basilisk :)

From this paper about black-headed gulls responding to a hedgehog:

The gull might peck the intruder, or strike it with its feet. Pecks were
  mostly directed at the hedgehog's head and might be delivered after a
  horizontal approach with the wings partly lifted.

Although not directed at the eyes specifically, they observed the birds directing attacks to the predator's head.
There's another paper (Ratner 1975) that also looks promising, but I'm on my home computer and can't access it right now

Any luck on the second paper? Thanks!

@DVK Sadly I don't have access to it at work either, sorry!


text:
What happens when we stretch?
From the wikipedia page on stretching:

Stretching is a form of physical exercise in which a specific muscle or tendon (or muscle group) is deliberately flexed or stretched in order to improve the muscle's felt elasticity and achieve comfortable muscle tone.

How is this improvement of elasticity achieved, at the physiological level?
Searching around on this topic brings a lot of articles about the types of stretches (e.g. again, wikipedia) and what are their benefits and disadvantages.
But how do they differ at the biological level?

here is a youtube video on how stretching work

Short answer: Due to habituation. You do something enough number of times, then that event ceases to be something new, and your body adapts to it.
In this case, you train your body to accept more risks allowing greater lengthening, before it begins to internally signal the muscles that an injury is incoming.
Read below for the slightly longer version:

The stretching of a muscle fiber begins with the sarcomere, the basic unit of contraction in the muscle fiber. As the sarcomere contracts, the area of overlap between the thick and thin myofilaments increases. As it stretches, this area of overlap decreases, allowing the muscle fiber to elongate. Once the muscle fiber is at its maximum resting length (all the sarcomeres are fully stretched), additional stretching places force on the surrounding connective tissue.
There are two kinds of muscle fibers: intrafusal muscle fibers and extrafusal muscle fibers. Extrafusil fibers are the ones that contain myofibrils. Intrafusal fibers are also called muscle spindles and lie parallel to the extrafusal fibers and are the primary proprioceptors in the muscle.
When the muscle is stretched, so is the muscle spindle. The muscle spindle records the change in length (and how fast) and sends signals to the spine which convey this information. This triggers the stretch reflex (also called the myotatic reflex) which attempts to resist the change in muscle length by causing the stretched muscle to contract. The more sudden the change in muscle length, the stronger the muscle contractions will be (plyometric, or "jump", training is based on this fact). This basic function of the muscle spindle helps to maintain muscle tone and to protect the body from injury.
One of the reasons for holding a stretch for a prolonged period of time is that as you hold the muscle in a stretched position, the muscle spindle habituates (becomes accustomed to the new length) and reduces its signaling. Gradually, you can train your stretch receptors to allow greater lengthening of the muscles.

You can read more about it (including the role of the lengthening reaction by the gogli tendon organs) here: http://people.bath.ac.uk/masrjb/Stretch/stretching_2.html#SEC13
It has also been published in a short section in this paper. I am sure you can follow the reference chain if you need to.
Smith, Craig A. "The warm-up procedure: to stretch or not to stretch. A brief review." Journal of Orthopaedic & Sports Physical Therapy 19.1 (1994): 12-17.


text:
Similarity between the human genome and archea genome in deep sea hydrothermal vents?
I'm trying to find some reference that shows what percentage of the human genome is similar to some organism from the domain Archea that lives  near or on deep sea hydrothermal vents.
Can someone points me out in the right direction?

The genomes are so different that it is pretty impossible to make any alignment expect for few very conserved region. I guess that you may be able to align histone genes eventually ribosomes (although they differ a lot). You may want to find these sequences and align them by yourself for training.

@Remi.b         I am curious, but would alignment of these genomes be done by some BLAST like program?

@Ro Siv Yes, exactly.

To achieve whole genome alignment between a genome as large as a human, and as small as an archea organism, is possible, but in the end, you'll have to strain your eyes to see patterns. The archea genome is about 1-5 million bp long, whereas the human genome is about 3.3 billion bp long (that's a thousand times too large).
In practice what this means is that you may or may not have small islands of similarity, in vast regions of emptiness.
Nevertheless, you can align whole genomes using the MUMer tool (http://mummer.sourceforge.net/). The white paper is here
Delcher, Arthur L., et al. "Alignment of whole genomes." Nucleic acids research 27.11 (1999): 2369-2376.
You might also be interested in reading this:
Wei, Liping, et al. "Comparative genomics approaches to study organism similarities and differences." Journal of biomedical informatics 35.2 (2002): 142-150.


text:
Extracting cell-free DNA
Has anybody had any success in extracting cell free DNA from plasma without using expensive kits? I've already spent a lot of my own blood trying to use standard phenol/chloroform methods with very minimal yield. I know it's possible that since I am not pregnant and don't have cancer (I hope) my blood may not have a large quantity of cell free DNA to begin with but I'm afraid to start using precious experimental samples until I'm confident I have a technique that will work.
For some background, cell free DNA is DNA that is circulating in the plasma (as opposed to the majority of DNA which is cellular). This has become an exciting new way to analyze fetal DNA from a mother's blood without doing a dangerous amniocentesis. There's also strong evidence that tumour DNA can also be found in some abundance this way (most studies seem to be with prostate cancer).
Qiagen has a kit for sale to extract this DNA but it is pretty expensive and I would still think that phenol/chloroform would give me a high (if less pure) yield. There is a paper in Cancer Biomarkers 2013 by Mazurek et al that claims that compares methods and shows a reasonable yield with this method but I haven't reproduced it. Has anybody here had success and if so what modifications did you use? Is it just time to move on to using samples that are more likely to have larger amounts of cell free DNA?

I know that if I inject plasmid DNA into a mouse tail vein with nothing to protect it, it's chopped up within 5 minutes. You sure the DNA is really naked DNA floating freely in the blood? There an awful lot of nucleases in there.

No, it is contained within microparticles or enveloped within a membrane as far as I understand but it is not cellular and can be found in plasma.

How about using a density gradient seperation to enrich your atarting material? Also, instead of precipitation, using silica microbeads to pull the dna after getting rid of the lipid membranes (in presence of appropriate nuclease inhibitor cocktail, of course!) might possibly increase your yield.

purpose of phenol/chlorophorm extraction, as I remember, is to precipitate/separate proteins, lipids and DNA. So if your dna is bound to proteins, you won't clear it

Maybe do a proteinase K digestion before your phenol chloroform extraction. That would destroy the nucleases. If you extract first, you'd probably have some nuclease carry over and damage your freshly unprotected DNA.

Is it worth comparing yields between serum and plasma ?

If you're worried about nucleases, try guanidinium thiocyanate with your extraction. I doubt that's your problem, though.

To note: I have no personal experience with this protocol. I am forwarding this paper, passed on to me by a colleague
There are many kits and methods available that don't rely on Qiagen. Or alternatively, if you ask them nicely, they do heavily discount their first kit you buy off them as a sort of trial (as they dont do samples).
However, to answer your primary question, are you adding a detergent and heat denaturing your samples first before phenol-chloroform (Triton/Heat/
Phenol)?

Blood samples for CFDNA
  analysis are collected into EDTA tubes and held at/or below RT
  (18 °C–22 °C) for no more than 2 h before separation of plasma.
  Plasma should be separated from whole blood samples by double
  centrifugation (800 g and 1600 g for 10 min, separately), avoiding
  leukocyte lysis. We prefer to isolate CFDNA immediately after plasma
  separation to minimize the effect of prolonged storage. Otherwise,
  samples should be aliquoted into small portions and stored at −70 °C
  before extraction because fragmentation of CFDNA might be caused by
  repeated freeze–thaw cycles
For the THP method, 500 μl of plasma/serum was mixed with 5 μl Triton X-100
  (Sigma-Aldrich, UK) and heat denatured at 98 °C for 5 min. Samples were placed on ice
  for 5 min, then extracted with an equal volume of phenol–chloroform–isoamyl alcohol
  (25:24:1, v:v:v) (Sigma-Aldrich, UK) and centrifuged for 10 min at 14,000 g. The
  aqueous phase was precipitated overnight with 1/10 volume of 3 M NaOAc and 2.5
  volume of 100% ethanol at −20 °C. The DNA pellet was washed with ethanol, air-dried
  and resuspended in 50 μl of ddH2O.
In the THP protocol, 98 °C incubation was used to inactivate
  PCR inhibitors and denature proteins in plasma/serum samples.
  Different times and temperatures were tested before choosing this.
  Since we had noticed substantial DNA contamination in SDS, Triton
  X-100 was used in the protein solubilization step. Direct conventional
  PCR was performed successfully on heat-denatured samples
  and able to detect 25 ng/ml DNA, indicating that this simple
  procedure offered sufficient protein digestion and inhibitor elimination
  for PCR analysis. The THP protocol gave a significantly
  higher yield of pure DNA solution for qPCR analysis than the
  QIAamp kit. The THP protocol showed high efficiency even with
  small DNA fragments as low as 100 bp.

You might want to read this:
Xue, Xiaoyan, et al. "Optimizing the yield and utility of circulating cell-free DNA from plasma and serum." Clinica Chimica Acta 404.2 (2009): 100-104.
A quick search of google scholar also revealed a few other papers you might be interested in:
Fleischhacker, Michael, et al. "Methods for isolation of cell-free plasma DNA strongly affect DNA yield." Clinica Chimica Acta 412.23 (2011): 2085-2088.
UPDATE:
After reading this (Fong, Siew Lee, et al. "Comparison of 7 methods for extracting cell-free DNA from serum samples of colorectal cancer patients." Clinical chemistry 55.3 (2009): 587-589.) which gives about 7 protocols (sadly without any references for some reason) I went hunting for  came up with this:

DNA was extracted from 1.0 ml plasma by phenol-chloroform method with slight modifications.
  One ml of plasma was treated with 1x SDS/Proteinase K solution (0.5 mg/ml) (1:1), incubated
  overnight at 56 °C followed by phenol-chloroform (4:1) treatment and centrifuged at 7 000 rpm.
  The upper layer was transferred into fresh 15 ml centrifuge tubes and the same step was repeated
  again; DNA was precipitated by adding glycogen (0.1 µg/µl), ammonium acetate (7.5 M) and
  absolute alcohol. DNA pellet was obtained by centrifuging at 7 000 rpm; was washed with 70%
  alcohol at 10 000 rpm, dried and finally dissolved in TE buffer at 56 °C and stored at -20 °C until
  further use.

You can read about the above method here: Singh, Deepika Misra1 Renu, and Monisha Banerjee. "A simple and reliable method of obtaining fetal DNA from maternal circulation; its accuracy and sensitivity."
Also, if its available to you, it might be worth it:
Gahan, Peter B., ed. Circulating Nucleic Acids in Early Diagnosis, Prognosis and Treatment Monitoring: An Introduction. Vol. 5. Springer, 2014.

Thanks for this! I have used essentially that second method except precipitated DNA with KCl instead of ammonium acetate... can't imagine that makes much of a difference. I also used SDS/Proteinase K but at a lower temperature. Because of my workflow I am unable to extract the DNA right away so I store the plasma at -80. I think the big thing though is that all of these papers use plasma that they expect will have cfDNA in it whereas I'm using control plasma so that may have the biggest impact on my yield I'm starting to suspect.

What temp is your proteinase K reaction? I've only ever seen it at 50 - 60 C.


text:
minimum ratio of the shortest isoform to the longest one derived from one same parent gene
I want to know what is the minimum percentage of the shortest isoform/transcript to the longest one that derived from the same parent gene? Does it have organism specificity? What is the function of the shortest isoform?
Because I want to filter the gtf file from cuffmerge. One consideration is that the very short isoform maybe artificial or random transcripts that should be filtered.
From this page 1, the CREB gene has been manually annotated and included a processed transcripts only 84bp, while the longest transcript of this gene is 8532bp. So the ratio is < 0.01; However, this transcript is not coding sequences. I also plot the minimum ratio distribution in human GENCODE v22 data.

1 vega.sanger.ac.uk/Homo_sapiens/Transcript/Summary?g=OTTHUMG00000097081;r=7:28724300-28726731;t=OTTHUMT00000327948
Best,
Pengcheng

maybe you are looking for proportion? That is ratio of isoform A to isoform B

thank you @aandreev, I have updated the content accordingly.

Splice product that short cannot be considered an isoform. If you are interested in splice products then you can have a look at miRNA arising from intron of the host gene.

@WYSIWYG thank you. I have disregarded the transcripts without annotated as "protein_coding". Yes, as you suggested, miRNA can be produced from intron, so the processed transcripts that detected in RNAseq potentially miRNA sequences.


text:
One Gene and many proteins
Imagine a gene with  $n$ exons and $m$ introns. How many proteins are possible from that gene? Would all the proteins be isoforms?

Is this a homework question or a assignment? If so, please tag it accordingly and tell us what you have done to solve this.

This question cannot be answered in its current form. How many of the exons contain a valid START codon? How many exons are in UTRs? How many actually code for protein and don't contain in-frame STOP codons? Please [edit] and make it more specific.

I might be wrong, but aren't numbers $n$ and $m$ are connected as $n=m+1$?
Answer seems to be combinatorial: how many combinations of $n$ objects can be assembled under certain restrictions? Namely, how many isoforms certain gene can have.
Restrictions include: how many exon-intron junctions on codon (or precisely between codons), how many exons are actually contain protein-coding sequence of mRNA (some exons are coding untranslated region, 3'- or 5'-UTR, for example), how certain gene processes alternative splicing.
So, as you can see, answer will highly depend on sequence of given gene. As far as I know, maximum number of isoforms is limited by 5, even though there are genes with hundreds of exons.