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Michaelis-Menten Kinetics: Does measuring apparent Km and Vmax take into account competing reactions?
I am learning about why it is important to measure Km and Vmax for each experimental setup because measuring the "apparent" Km and Vmax includes enzyme inhibitions of which one might not be aware.
However, what if there are entire reactions going on that are consuming the product or substrate that are not known? Are these accurately aggregated into the Km and Vmax measurements?
Here are some of the links that I read through:
http://www.bio.upenn.edu/media/pdf/courses/F02/BIOL202/Handouts/BIOL202.ApparentKmandVmax.pdf
http://en.wikipedia.org/wiki/Enzyme_inhibitor

Obviously no. If you read any text describing the derivation of the equation you will see that the reaction is considered in isolation. How can it take into account the unknown?

Have you heard of something known as "Occam's Razor" ?
It says when you have multiple possible explanations/hypotheses then select the one which is simplest (i.e least number of assumptions)
Same with mathematical models. Chemical kinetics models usually assume first order unless there is some evidence against it. Similarly, for enzyme kinetics, as long as you don't think there is something anomalous going on, use Michaelis-Menten model.

What if there are entire reactions going on that are consuming the
  product or substrate that are not known

Many physiological enzyme catalysed reaction are not Michaelis-Menten. If you happen to have an insight about it or if the simulations dont match the experimental data, you can revise the model.
However, Michaelis-Menten model for for competitive inhibition exists, as you yourself indicate in the link.

As Arthur Kornberg said: "Don't waste clean thinking on dirty enzymes."

Discovering an assay for a biological event in a cell-free extract opens the way to its molecular resolution and reconstitution (Commandment I). Trying to devise a mechanism with a crude extract, even with ingenious experiments, is generally a waste of effort. An extract is too dirty; purification is the only way to go.

Added in response to a comment from Dexter:
Kornberg was quoting Efraim Racker. And in any case my original link is dead.

+1 for awesome quote. But original quote is by Efraim Racker -- http://jb.asm.org/content/182/13/3613#sec-4

Thanks, I didn't know that. My link appears to be dead so yours is an excellent replacement.

The answer is no really, but some variants might allow you to study inhibition.
Michaelis Menten kinetics are experiments which try to characterize the catalysis characteristics of a reaction but to do so the numbers are obtained at concentrations of substrate that you dont find in a cell.
Vmax is defined as the maximum rate at which the reaction is run in question.  To measure this the substrate concentration is also maximal - the binding sites of enzymes must be fully occupied during the entire experiment. So by definition there is no competitive reaction possible here.
Km is a binding constant obtained where the reaction rate is found to be half Vmax.  This definition is free of any competing reactions.  You can use Michaelis Menten kinetics to study competitive inhibition though and by comparing a reaction rate and comparing it to the reaction rate (called Vo) you can derive a Ki which gives you how much your inhibitor has affected substrate binding.


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What's a good and reliable database on the pharmacodynamic and pharmacokinetic data of drugs both approved and unapproved?
What's a good and reliable database for the pharmacodynamic and pharmacokinetic data of drugs both approved (in the US and elsewhere) and unapproved?

For approved drugs, the Drugs @FDA database at CDER (Center for Drug Evaluation and Research) aggregates labeling information (which includes at least a cursory PD explanation and a listing of or reference to common PK data for the substance) for most modern drugs.
Unapproved drugs may be a bit trickier to find out about, but the FDA maintains a list of contract research organizations (CROs) who are running trials here, and the actual trials can be found on http://www.clinicaltrials.gov/, which will have citations for the papers that have been published.
Drugs in their earliest stages of development, i.e., internal to a pharma company, are likely to be kept pretty tightly under wraps.
I don't know enough about the governing bodies in other countries to know where any international data would be, but I assume you can find published papers for substances in those countries as well.

Where exactly on this site do you find the PD and PK data? I looked up sertraline all I got was these sort of pages...http://www.accessdata.fda.gov/scripts/cder/drugsatfda/index.cfm?fuseaction=Search.Overview

If you go to the brand for the drug (Zoloft), click on the pill form, and then click on the "label data", it will come up.  On the main screen with the brand drugs, it also has a link that says "label available"  (they aren't labels any longer, but those inserts that manufacturers put in, but they retain the old name).

@BrentonHorne The generics don't need labeling info, only bioequivalence, so I think the information for PK is retained with the original IND number.

If you haven't already, I'd recommend volunteering in a university lab, because you clearly have a strong passion for this with all of your questions!

Before I asked this question I knew of a website that provides reliable (from the US government) pharmacodynamic and pharmacokinetic data for FDA-approved drugs: DailyMed. I didn't accept it as an answer because of the fact that it is just FDA-approved drugs but I'm interested in other drugs like moclobemide, tianeptine, piracetam, aniracetam, nefopam, flupirtine and levomepromazine too.

source: Stack Exchange

license: Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/

sort: votes


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What are polytene bands, and why are they there?
Drosophila polytene chromosomes have been particularly useful in genetic research, as it made cytogenetic gene mapping possible with very little effort. This was primarily accomplished due to polytene bands, which are naturally-occuring (no stain) fixed bands seen along the chromosomes via microscopy.
We have been exploiting these bands for decades, but it seems as though their function isn't quite clear.
What is the cause of the visible polytene bands, and what function do they serve?

well, Wikipedia says that they are formed by numerous rounds of replication without cell division, and their function may be in gene dosage [but this is not really clear].  However, this is all without proper references, so you should probably wait until someone comes along with citations to verify.

This review has more information than you will ever want to know.

all_licenses: Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/


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How is Hypertonic Urine Produced in a Mammalian Kidney?
I know that the ascending loop of Henle is impermeable to water and ions, and, by end of the ascending limb, the osmolarity of the tubular fluid is very low due to the active transport of ions out (e.g, sodium, potassium, chloride).
However, what if the person had to produce hypertonic urine due to dehydration or some other circumstance? Would the increase in osmolarity be done through a decrease in removal of ions in the descending limb, or has the distal tubule and collecting ducts got something to do with it?

aquaporins in the distal tubule absorb water. they are controlled by vasopressin (anti-diuretic hormone)

The process of urine concentration in mammals occurs due to the interaction of the loop of Henle and its nearby collecting tubule. When someone is dehydrated, the kidney will aim to a maximal water reabsorption.
To concentrate urine, a maximal amount of ions will be reabsorbed. This is to maximalize the concentration gradient for water. However, the main driving force for water reabsorption is the urea concentration gradient (created by the loop of Henle). Then aquaporins will allow water to flow from the collecting tubule to the nearby medulla because of the presence of the neighboring loop of Henle. It is this proximity between the loop of Henle and the collecting tubule that allows such a high efficiency for water reabsorption.
Concentrating urine is not about leaving more ions in the water, it is about reabsorbing a maximal amount of water.


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What factors govern the variable age of onset in Huntington's Disease?
"Huntington's disease (HD) is a neurodegenerative genetic disorder that affects muscle coordination and leads to cognitive decline and psychiatric problems." As we all know, this genetic disease exhibits a generally late age of onset. It has been shown that a increase in the number of CAG repeats in the hungtingtin gene is negatively correlated to the age of onset of the disease. However, patients having a  particular number of CAG repeats still have an extremely variable age of onset, which leads to believe that other factors are contributing to the Huntington phenotype.(reference)
 (reference)
What other genetic or environmental factors have been shown to vary the age of onset of Huntington's disease?

References
Andrew, S. E. et al. The relationship between trinucleotide (CAG) repeat length and clinical features of Huntington’s disease. Nature Genetics 4, 398–403 (1993).

There is quite a bit of data available from Venezuelan HD families, but they haven't identified the factors themselves. From the abstract of the paper, emphasis mine:

Analysis of the 83 kindreds that comprise the Venezuelan HD kindreds
  demonstrates that residual variability in age of onset has both
  genetic and environmental components. We created a residual age of
  onset phenotype from a regression analysis of the log of age of onset
  on repeat length. Familial correlations (correlation +/- SE) were
  estimated for sibling (0.40 +/- 0.09), parent-offspring (0.10 +/-
  0.11), avuncular (0.07 +/- 0.11), and cousin (0.15 +/- 0.10) pairs, suggesting a familial origin for the residual variance in onset*. By
  using a variance-components approach with all available familial
  relationships, the additive genetic heritability of this residual age
  of onset trait is 38%. A model, including shared sibling environmental
  effects, estimated the components of additive genetic (0.37), shared
  environment (0.22), and nonshared environment (0.41) variances,
  confirming that approximately 40% of the variance remaining in onset
  age is attributable to genes other than the HD gene and 60% is
  environmental.

*This paper also suggests the same, with a smaller (but different) samples.
A recent study of an Italian sample suggests that a polymorphism in Atg7 (autophagy-related gene) is a modifier of age-of-onset, but this result does not generalize to a European cohort.

What is apparently not linked to age of onset in HD? β-defensin copy number variation.
Also, CTCF doesn't appear to be linked to the CAG repeat instability (mutations in CTCF have previously implicated in triplet repeat instability).
Mutations in PGC-1α, mitochondrial regulator, were previously associated with age of onset, but a more recent study suggests that this might have been due to stratification of the sample.
Similarly, a polymorphism in HAP-1 was previously associated with age of onset, but a subsequent study found no evidence of association.

Altogether, these results suggest that population stratification needs to be taken into account when evaluating associations between age of onset and genetic polymorphisms.  It is also possible that there are population-specific genetic factors that affect age of onset.


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What gives observable light its colors?
I know that difference between different colors of light is difference between their wave length but I don't know what gives beautiful colors (like rainbow colors) to different wave length of observable light?
What makes our eyes to see different wavelengths of light as different colors?

Remark:
I've meet someone which he was color blind, he was not able to see rainbow colors.
So the process that gives color to light must be in our eyes and brain.

This is not a physics question, but a biology question.  Please ask on biology.se instead.

Basically cone-cells in the eye detect colours and consequently send an impulse to the brain.

Our eyes detect light waves of different wavelengths, which we then perceive using our brain as colour.

@Chris: This really falls in the gap between physics and biology; I don't see it as belonging clearly to either. Is there a bio-physics.se yet?

Related to http://physics.stackexchange.com/q/48224/11062 and its answers there.

i think the clearest understanding you could have here is that we see these colors as an evolutionary adaptation (for example, we see a wider range of green vs other colors). the reason we see within this particular spectrum is because it's the spectrum of light which survives Earth's atmosphere on its way to our eyes, so it is evolutionarily favored (in the sense that we get more information from our environment by being able to detect light in this range)

Achmet, I've linked one duplicate of your question, but if you search this site for "eye color cones" or something similar you'll find many related question.

This is a philosophical question rather than physical. I believe it is off-topic here. I suggest you to look up for for qualia. http://en.wikipedia.org/wiki/Qualia http://plato.stanford.edu/entries/qualia/

@Anixx: You have been told before: Philosophy-like tags like 'metaphysics' tag are not allowed, cf. e.g. this and this meta Phys.SE post.

Three different types of cones in the eye are sensitive to three different, contiguous and slightly overlapping, sections of the visible spectrum. The nerve signals from these three different types of cones are interpreted by different sections of the brain as cyan, magenta, and yellow, from which all the discernible colours are composed. This forms the familiar Colour Triangle.
The sides of the Colour Triangle bulge slightly (I believe) because of the slight overlap in the sensitivity ranges of the three cone types.
Colour blindness occurs when someone is born with only two (or rarely one or none) different types of cones, instead of the usual three. This causes an inability to detect one of the thee sub-spectra.

you should add a sentence that addresses the double value :both the frequency spectrum is seen correctly and the mixture of three frequencies can give the same color response even though the "clean" frequency is missing.


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PEG-silane treatment: why incubate for 18 hours at 60 degrees Celsius?
I am conducting a biochemistry-related experiment and I have been unable to understand a step which is commonly performed.
My aim in this step is to apply a PEG (Polyethylene glycol) silane layer.
After immersing indium tin oxide (ITO) slides in a PEG-concentration, the slides are incubated for 18 hours at a temperature of 60 degrees Celsius.
Can you tell me why the incubation is performed? And why for 18 hours and at 60 degrees Celsius?
Thanks a lot in advance!!

please clarify the experimental aim and procedure. And please expand the abbreviations at least once.

plus.. this question doesn't come under the domain of biology. try chemistry SE if your actual question doesn't involve any biological principle

The binding of proteins (and cells) to glass (or silicon) surfaces can be prevented by coating the glass with polyethylene glycol (PEG) groups. PEG-silane is a reagent used to create this coating.
PEG-silane (the image shows a methoxy- version) (image taken from here; no connection)  will coat glass surfaces because the silane portion (right hand end of the structure shown) will react with -OH groups on the glass surface.
I can't help with the part of the question about time and temperature of incubation, but it seems longer and hotter than protocols I have seen. Perhaps the indium tin oxide (ITO) coating on the slides has something to do with it?

Thanks a lot for your response Alan! What I do not understand yet from your answer is: where do the OH-groups on the glass surface come from?

Google Images: glass chemical structure

please go through the journal: S. Jo, K. Park, Biomaterials 21 (2000) 605-616.
The time and temperature probably helps to hydrolyze, formation of hydrogen bond and covalent bond formation. Hope this paper will help you.
Good luck!


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Copper for cell incubator to prevent contamination
For some reason the lab seems to have a problem with contamination every so often.  It's virtually impossible to prevent bacteria, viruses, fungi, etc. from getting into the incubator every time you open the door.  Our incubator is pretty old, we don't have one of the new fancier versions that's made with pure copper inside, which effectively kills microorganisms.
Would it be a good idea to buy a copper plate and place it on the shelf, on which we could put our culture flasks?  Or would it disrupt the flow of heat/CO2 too much?  Would it be better to buy copper foil and wrap the shelves in it, then poke holes into it where all the holes are in the shelf?
We use good cell culturing practices; I don't contamination is happening because people don't know what they're doing.  It just happens over time with enough people working in a cell culture room.  Could copper in the incubator help?  We probably don't have the money to buy a brand new incubator that's made out of copper, so would this be a viable, economic alternative?

First thing I'd do is replace the HEPA filter. A copper plate/foil may help, and it certainly won't disrupt the flow of heat, as copper is incredibly conductive (that's why they make electrical wires out of it), and poking holes would help with the airflow, but a new filter will probably make the most difference. I've never used a full-copper incubator, and the few serious contaminations I've had to deal with were related to the HEPA filter and poor TC practices.
If you haven't already, I'd institute a good routine cleaning program as well. Change the water bath each week (make sure you're adding fungicide to the water), and wipe down the whole incubator with bleach followed by an alcohol rinse each month. Make sure you clean all the bleach off, as it corrodes metal if left on very long. At the same time, clean and autoclave all of the removable components like shelves, rails, and water pans.
Good luck!


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How are new people created from the DNA of an aged person. i.e. Why are we young?
The question "why do we age" has been asked numerous times. But why are we young? The cells of the adult human being are an age (time>0), but how can old cells create new cells that are younger than themselves?
I.e, how are young people made from the cells of old people?
I realize that senescence, the act of a cell aging, has to do with the shortening of telomerase over time which puts a limit (the Hayflick limit) on the life of an autosomal cell. So in theory, because cells only make direct copies of themselves but the telomerase shortens with every division, each new daughter pair would be as old as the parent and would continue aging. But how come new people start senescence at time t=0?

Telomeres shorten with every cell division. Telomerase is an enzyme that adds extra copies to the ends of telomeres, extending a cell's lifespan. The assumption that telomeres only ever get shorter is incorrect (though that doesn't invalidate the rest of your question).

Gametes (sperm and ovum), which fuse to form a zygote, arise from germ cells (spermatogonia and oogonia). Germ cells, like stem cells, are maintained carefully i.e the genome is preserved and transposition/recombination events are tightly controlled via different mechanisms.
So these germ cells don't have shortened telomeres. Also, during early embryonic development the future germ cells quickly migrate to an end of embryo and remain there while the rest of the body takes shape.
I'm not sure if it has been demonstrated in an article or not but once I happened to talk to Dr Wakayama (who cloned a mouse frozen for 16 years) and got to know that animals cloned by the process of Somatic Cell Nuclear Transfer have reduced lifespan. (SCNT: the nucleus of a somatic cell is transferred to an ovum and triggered to form zygote).
So, to conclude, if germ cells were not protected from mutations and deleterious recombinations, lifespan would have been reduced with every generation.

I guess it would be good if someone could give a word about epigenetics too! Gametes do not carry the same epigenetics modifications than cells of old people.

That is a good point. It is observed (to an extent) that transfer of somatic nucleus to egg cytoplasm causes epigenetic reprogramming. However, I am sure that the old epigenetic marks are not completely erased.

And I guess it is intuitive that no two cell types have same epigenetic marks.

Oocytes have the special purpose of resetting, reprogramming epigenetics based on many physiological factors of the mother. That epigenetics are reprogrammed by the oocyte accounts for many of the differences between twins.

It could be interesting if a human could use its own germ cells to keep creating "young" cells for itself, and thus never age.


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Two brown-haired people have two children, P and Q. P has blond hair. Q has brown hair. What is the probability that Q is heterozygous?
Two brown-haired people have two children, P and Q. P has blond hair. We therefore believe that each parent is heterozygous and that blond hair is a recessive trait.
Q has brown hair. What is the probability that Q is heterozygous?
My answer:  2/3.  There are four possibilities Bb, bB, BB,and bb. Before observing the color of Q's hair, we assign equal chance to each.  We eliminate bb as a possibility.  The chance that it is bB or Bb and not BB is 2/3.
A Biology PhD disagrees with me and says that the probability is 1/2. Who is right?

now ask that PhD to solve the Monte hall problem.

@Memming that's http://en.wikipedia.org/wiki/Monty_Hall_problem

I am familiar with the Monty Hall problem.

Assumptions: Blonde hair is Homozygous Recessive and that the traits are strictly Mendelian.
The parental generation must be both heterozygotes as at least one child is Blonde (bb). So your cross is Bb x Bb.
Your square is going to look like this:
           _B_            _b_

_B_         BB           Bb

_b_         bB           bb

So of the question is:

What is the probability that Q is heterozygous?

The PhD is INCORRECT. They probably just made a simple Punnet Square in their head and forgot the caveat that you have to exclude "bb" because Q is not Blonde. The possible allele combinations for non-Blonde offspring from a Heterozygotic cross are only BB, bB, Bb.
Therefor the probability that Q is Heterozygous is indeed 2/3 (bB, Bb).
It's not uncommon for PhD students - who are often deep into their own research - to gloss over details and possibly give incorrect answers because the information hasn't been relevant to them for years (most traits aren't Mendelian, strictly recessive or dominant, and rarely involve a single allele), so give them some slack unless they were being a jerk about it. =)

Just to complete the answer, this essentially is an application of Bayes' theorem

I worked it out that way, too.  But adding another layer of mathematics isn't going to prove anything to someone who disagrees with the simple way of seeing things.

Then you have one more argument: this was probably a high school homework, so it has to use what kids learn about Mendelian heredity, not something deeper and more elaborate.


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Separating DNA Fragments by Gel Electrophoresis. Are all the strands for one size the same?
My apologies if my question is too basic, and please point me to a more appropriate forum.  I am reading the textbook "Essential Cell Biology" by Alberts et al, and am consulting other sources as well.  I am reading Chapter 10 about DNA technology and have read how DNA is separated into strands of different lengths by restriction enzymes.  The different size strands can then by separated by Gel Electrophoresis.  I believe I understand these basics. My question is:
Do all strands of the same length have the same sequence of base pairs?  I wouldn't think      so, and this would be a problem in the next step, determining the sequence of one size fragment by  using special base pairs that are lacking a hydroxyl group. Isn't it possible I could have two strands that are 50 base pairs long (for example) that have the same beginning and same end and are thus cut by the restriction enzymes to a length of 50, but are most assuredly not the same within their sequence
The text says that once the different size strands are separated by electrophoresis, you can just cut out one of the bands and work with that.  That makes sense if you are guaranteed to only have 1 strand at that level of the gel agar or if the multiple strands are all the same exact sequence.
If you read this far, thank you.  I think more generally I need to know how many of these segments are produced when you use restriction enzymes and how long the average one is. And is there any guarantee for one chromosone (or all 46 chrmosones) having all the segments be unique sizes (I wouldn't think so)?
And even more generally :), I need additional sources that explain DNA sequencing in simple to understand terms.
Thanks,
Dave

You are correct: strands of the same length do not necessarily have the same sequence. The separation of DNA in gel electrophoresis is purely based on the hydrodynamic size.
The number of fragments produced by restriction enzyme digestion depends on the number of sites for that particular enzyme in your input DNA.  Restriction enzymes only cut at specific nucleotide sequences.  There is no "typical length" of fragment produced for this reason.
The idea of separating DNA on the basis of an RE digest is that a mutation that changes the sequence where the RE binds and cuts will reduce the number of fragments because the RE can no longer bind and cut at that site: 2 smaller fragments will now appear as one large fragment.  It will be visible on the gel because of the difference in number and size of fragments.
Typically, when running a gel, you are using DNA that has been selectively amplified to bias the readout to a particular (known) region of the chromosome. This electrophoretic approach works because you're not looking at all 46 chromosomes at once.
As far as sequencing is concerned, I'd recommend starting with Wikipedia.  Sequencing techniques are changing rather rapidly, so there's not one good source of which I am aware. To begin with, you can read about Sanger sequencing method. If you understand PCR then you won't find it hard to understand.

Thank you for your quick response.  I'm still not sure I have an answer to my question.  Perhaps the question is just too obvious and the answer is in front of my face.  If as you say, "strands of the same length do not necessarily have the same sequence" how does one separate them for subsequent analysis to specify that actual base pair pattern? If I understand the book correctly, strands will fall in the gel according to the number of base pairs they have.  Thus two completely different strands that are both 50 base pairs long will fall to the same height.

If I then try to figure out the sequence of this "strand", that is really two strands (or three, four, etc.) won't I just have a mess?

Yes, differentiating the strands is not possible on this basis: restriction enzymes can only be used to look at changes at a particular site, but it doesn't tell you what else is going on, or what the change was in the first place.  The sequencing method that you want to take a look at is Sanger sequencing...things have progressed since, but that is how you'd actually use electrophoresis to sequence.

All is dependent on how much you are sure about your experimental result. If you are very sure as in case of a plasmid digestion or a gene specific PCR then the band that you are getting will most likely be the strand of your interest.
However, when you are unsure about the experimental outcome then there is no better way to check what a band is composed of, other than sequencing. [in most next-gen sequencing experiments, the "libraries" are comprised of bands of sheared DNA migrating together, lets say at around 200bp. In this case the aim is to sequence the unknown]

The answer to the part of your question concerned with average lengths of restriction fragments is: if the DNA molecule being digested is of random sequence, and is 50% GC/ 50% AT, then the probability of finding any given short sequence at any position is 1/4N where N is the length of the short sequence. So, for example for a restriction enzyme with a 4-base recognition sequence such as AluI (AGCT) the probability is 1/256 and so the average length of an AluI fragment would be 256 bp. For an enzyme with a 6-base recognition sequence such as HindIII (AAGCTT) the corresponding number is 4096 bp. Please note that this is the average length, and is not an 'expected' length.
The answer to the part of your question about fragments of the same size co-migrating, how would you purify them on a gel for further analysis is trickier. I think the first thing to say is that you wouldn't do this for sequencing, and this is reflected in the various answers you have already received - sequencing no longer requires this sort of approach.
If you were forced into this position for some reason then I can think of two things that could help: the first option would be to run the analysis on an acrylamide gel when you can get much better resolution of different fragments (all 50 bp fragments are not the same molecular weight), but this is really only useful for small fragments (100 bp or less). However the most likely way out of the co-migration problem would be to purify the two comigrating fragments as a mixture and then ligate them into a vector whereupon each recombinant would carry one or other of the two fragments. This is really going back to the original reason why molecular cloning revolutionised things - it allows the 'biological' purification of DNA fragments followed by production of these fragments in large amounts.
You mention "special base pairs that are lacking a hydroxyl group" so clearly you are thinking in terms of Sanger (dideoxy-) sequencing, in which case you could clone directly into an M13 phage vector so that you could make ssDNA for direct sequencing. By sequencing a number of clones you would find two sequences, one for each fragment. You would still have to find a way to decide which one was the fragment that you were actually interested in, however.
Like I say, things just aren't done in this way anymore.

As I understand, you are confused how one would be sure that a band in a gel is the sequence one is looking for, seeing as sequences can be different and still have the same length (e.g. ACTTAT is the same length as GGCCGT but entirely different). So your actual question might rather be something like "How does one ascertain that a gel band contains only the sequence one is looking for?"
It is true that in practice, restricting a long sequence can lead to similarly long segments which are not the same sequence. It is indeed possible to cut out a small square from the gel which contains the band, and then dissolve the gel and extract the genetic material. Sequencing the result would give you one of two outcomes: 1) If the band contained only your desired sequence, your sequencing technique should render exactly that sequence as a result. 2) If the band contained more than one different sequences of the same length, your sequencing method will probably leave you with ambiguous results.
So sequencing would probably only confirm whether or not the band contained a single distinct sequence. It would not be useful as a method of separating the sequence you want for future experiments from any other sequence of the same length. One method to do this could be PCR (polymerase chain reaction), a procedure that produces vast numbers (billions) of copies of a specific target sequence. If a PCR was carried out to amplify the target sequence contained in the gel band, any other sequences of the same length would not have any copies produced. They would still be present, but being vastly outnumbered by the sequence relevant for your experiments should prevent the other sequences from having any significant effect.
However, as dd3 noted, you would not normally run a the result of a restriction on a gel and then try to separate your target sequence from the rest. Rather, you would simply use the PCR method described above directly after using the restriction enzymes, and then run the result on a gel. You would then get a very strong band for the sequence you were looking for and much weaker bands for everything else.


text:
What is the function of nodes of Ranvier in axons?
In a neuroscience class I'm taking, it was explained that myelin covers axons in sections, the uncovered sections are called nodes of Ranvier, and signals propagate much faster in the covered sections.
But if it is faster for signals to propagate along myelinated regions of the axon, why doesn't the entire axon get covered by myelin? Wouldn't that make it faster?

Every signal rapidly fades out with distance if it is not amplified. So if the whole axon were covered with myelin, action potentials wouldn't reach their target. Each Ranvier node can be seen as an active signal repeater.

Can you reference any original research paper about this "distance" that every myelinated portion should have?
In another question I just posted ( https://biology.stackexchange.com/questions/67879/why-myelin-sheat-does-not-cover-the-whole-axon/67882#67882) @Kesley R. told me that the optimal myelin sheat lenght is about 1 μm.
But wouldn't it depend also on diameter of the actual axon and other physical properties?

Action potentials arise and propagate by membrane depolarization.
A myelin sheath is present in most neurons and is responsible for insulating the membrane against ions. The function of the myelin sheath is to prevent leakage of charge, by effectively restricting ion channels only at certain regions called Nodes of Ranvier. This ensures that depolarization at one node is effectively equivalent to depolarization at another node, which initiates an action potential at the later node and as a result the action potential effectively "jumps" from node to node - this is called saltatory conduction and it speeds up the signals.
In axons without a myelin sheath, the signal does (passively) travel the full length of the axon.


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How to know if a woman is fertile without actually making her pregnant?
How to know if a human female is fertile (can give birth to child in future) without making her pregnant? Any signs, tests?

Menstruation would be an obvious one

If you are talking potential for fertility. There are a few...

Having regular periods
Healthy weight. Extremely thin women tend to stop ovulating. Obesity also impairs fertility by altering hormone levels.
Whether she has been using birth control (i.e., "the pill") in the recent past. It takes a while for the body to get back to normal.
Smoking impairs fertility
stress

Source
If you are talking about immediate fertility...
While I would not rely on them for the purposes of birth control there are several observable physiological changes that correlate with ovulation and fertility. These include

body temperature. There are patterns of elevated body temp associated with ovulation.
cervical mucous. The mucous gets "slippery and stretchy".
the cervix itself becomes less firm and slightly open during ovulation.

Source
If we are talking about anatomical cues that are proxy's for fertility, there are clues based on the concept of sexual selection.

Ample breasts. Indication of potential mammary tissue and ability to suckle child.
Wider hips. Indication of ability to deliver child with minimal complications.

Note that these cues can be deceptive. Fat tissue may be deposited in both locations to imitate the fertility cues. Source

Of course. There's several fertility tests available to determine the quality and quantity of egg reserve, and whether or not the there are abnormalities in the uterus.
However, some of them are invasive procedures, and all of them should be performed by a professional. I doubt the layperson would know what they're looking at on an Ovarian Ultrasound.
If you're asking whether there's an "easy" way that doesn't involve a trip to a doctor - then I would say 'no.' Casual inspection would only reveal any abnormalities in the vagina and the opening of the cervix, and hormone tests are only partial indicators.
Then again, if a woman has reached physical adulthood and is under the age of 38 then the chances of being infertile (or have difficulty conceiving) are quite low - about 3% to 10%. Infertility is usually much more of a concern to women who wait until after the age of 35 to have children, as there is a definite biological window (the number of eggs the woman was born with) to consider that starts to close as a woman nears her 40's.
It's also important to note that trouble conceiving can stem from either partner, and there's about a 40%-50% chance in a couple trying to conceive the male has an 'issue' and the woman is perfectly healthy.


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How does geography affect morphological features of the human body
I've seen many times how a person born in one place, goes to another country for a long time, and then they start looking more like the people there, but I never found out how it works.
This report claims that second generation Japanese, born in the US are taller and heavier than those living in their native lands (there had been no intermingling of races).

To what extent do the genetic/environmental factors control human morphology ?
What factors apart from the diet can bring about such morphological differences ?

I think this needs to be referenced as an actual phenomenon in order to be answerable.

I'm searching for any relevant research documents right now, and I only find blog posts of people wondering the same thing, but the answers are only based on guesses.

@user1306322: added a reference for you.. co-incidentally someone told me about this just few hours ago :)

I think that particular instance probably reflects dietary changes more than anything else.

Geography does not typically affect the morphology of the human body.
In certain special cases, there are physiological adaptations, the most obvious being tanning and acclimatization to high altitude, but these aren't changes in body morphology.
So why the difference in children of immigrants?  This is because diet, nutrition, and disease have quite strong effects on children's growth. Kids growing up in a healthier environment with more food get bigger, and those are linked to the wealth of the community where you live.

+1 There is also cause-and-effect relation involved here: immigrants are not a random sample from the population of their original countries. One may argue about whether this translates into educationald and professional achievement, which may affect physical appearance... but teh comparisons between the average immigrants and non-immigrants are likely skewed.

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


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Can Infant Crocodiles digest Bread?
I'm just curious as to whether crocodiles and if you require specifics, saltwater crocodiles, could eat bread as infants. I was watching Lake Placid and I was curious as to whether infant saltwater crocodiles could even digest bread.

From a quick search it seems that amylase production in reptiles is highly variable and is generally a function of the eating habits. According to that logic crocodiles shouldnt produce amylase. Therefore I shall guess that crocs cant digest bread

This question seems more suitable for Pets-SE

If @WYSIWYG could convert his comment in an answer I think this is an OK question. I haven't seen much folks holding a croc as pet ;)


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How long does a tan last?
How long do melanocytes and melanosomes continue to protect DNA after UV exposure? Basically, if skin is tanned, then over the next month it is shed, will melanocytes continue to produce high levels of melanin to keep the new skin tanned without requiring more UV exposure? The cells react by producing more melanin after exposure but will they continue to do this in new skin cells?

The tanning response to UV occurs in two phases:

The immediate response begins at exposure to UV-A, reaches a max at 1-2 hours, and fades between 3 and 24 hours.
This article speculates that this is due to the redistribution of melanosomes rather than additional melanin production.
The delayed response is more durable, occurring by repeated exposure to UV-B, as well as as UV-A and visible light. "It is a gradual process in which the skin starts darkening 48–72 h after irradiation, reaches a maximum ∼3 wk after exposure, and the skin does not return to its original melanin content until ∼8–10 months later."
This source says this is due to additional melanocytes, melanosomes, and more melanin, in general.

Both processes are influenced by genetic factors.
Source for the initial points, which also contains interesting details on the enzymes involved in these processes.

Thanks for the resources, I'll need some time to read them :-D

Not a problem - it was interesting. I might start with the one source I linked to twice in the body of the answer. Although it's older, it is also not written by Avon scientists, and is less likely to be biased.


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How does sugar enter neurons if they don't use insulin?
I heard somewhere that as opposed to other cells, neurons do not use insulin to get their sugar supply.
Why is that?
What is the alternative mechanism? I assume sugar can't just enter the cell without some kind of help from a protein, is that true?

Insulin is not directly involved in sugar uptake. It accelerates sugar uptake by upregulating glucose transporters and activating glycogenesis.
Neurons have many glucose transporters including GLUT1 and GLUT3 which are responsible for glucose uptake.
Moreover, insulin being an endocrine molecule has effects throughout the body including neurons. So neurons need not secrete insulin.

It's important to have vital cells not require insulin for glucose uptake. This includes organs such as the brain or cells such as red blood cells. They get glucose by diffusion which only stops if glucose levels get ridiculously low, which is most commonly seen in a diabetic that's taken too much insulin.


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Why do animals sleep
Animals spend a good portion of every day doing nothing, and it seems like that time could be better spent hunting, searching for mates, etc. Also, animals are not aware during sleep, so it would be easier for a predator to attack them. Why is sleep required for animals?

different animals have different sleep patterns.. can you clarify which animals are you talking about

In humans, sleep is required for memory consolidation.  You might also be interested in this question (re: the predator question): http://biology.stackexchange.com/questions/7616/do-dolphins-really-sleep

Just to add to this conversation, sleep also has significant influence on the immune system function as discussed in this paper (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3256323/)


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Results of a complete DNA sequencing - are they 100% reusable?
Is that correct that a complete DNA sequencing (the whole genome) need only to be done once (per person)?
After that is done, it the complete genome can be stored and once the new genes (and their purposes are confirmed) are discovered, the scientist can simply go back to a previously sequenced data and to find it this particular person has this new gene or not.

A single run of sequencing won't cover the entire genome, in most cases. That's why they do multiple rounds in order to increase horizontal (covering more regions of genome) and vertical (a.k.a depth; more reads per locus so that you can be more confident) coverage.
Edit by dd3:
In the cases where "a new gene is discovered", i.e. genome reannotation, it is possible to go back and look at previously stored sequence data to find out what alleles a person has (everyone has every gene, unless they have a deletion that spans the full length of the gene). This is a bioinformatics project, and if you are interested in learning more, I would recommend searching for recent publications on genome reannotation and remapping of sequence reads.
Whole genome sequencing is generally not recommended for prognosis of a specific disease like breast cancer. The common approach is to do a PCR of the biomarker loci and if necessary do a Sanger sequencing. However, if you have the money you can get your whole genome sequencing done :P
The number of runs required to increase the depth varies for different genomic regions. This article says that CpG islands require more runs. According to this article, the depth should be >3 per genotype marker.

@WYSIWIG... Thank you. But how does it work in practice. Let's say someone wants to be tested for probalility of occuring a breast cancer. In that case - will only a particular region of genome be sequenced to find if a BRCA1 gene is ok? Please excusing me for a naive questions - but how many runs (approximately) are required to sequence the whole human genome?

edited the answer.. the comment was getting too long.. :)

for many of the technologies in use today, 30x coverage can still be problematic.  Should mention that the sequencers have biases which systematically give hard to interpret or easily misinterpretable data.  but given this, the data could and should be reused if its a whole genome sequence.

excuse me @shigeta, what is a "30x coverage"?  Thank you.

coverage is an estimate of the number of times each individual base in the target sequence has been sequenced.  so 30x coverage means you would have produced an assembled sequence where 30 sequencer outputs would contain an average base.  the coverage needs to be so deep because the short sequences from the sequencer (a few hundred bases) there is some uncertainty in each sequence, errors in sequencing, and  the resulting difficulty placing any given sequence. this paper might help:http://www.ncbi.nlm.nih.gov/pubmed/23393025

@Mitten: Coverage (vertical coverage) as I mentioned previously, is number of reads that map to a region of the genome. We generally look at the average coverage throughout the genome.

To your point, fast forward to today, whole genome sequencing is more affordable  costing about $900 (without compromising on privacy) for the following 130x coverage for the Exome, 30x for the rest of the Genome. The cost of Genome Long Reads is still very expensive.

Is that correct that a complete DNA sequencing (the whole genome) need
  only to be done once (per person)?

Likely, yes.  But I think you vastly overestimate how often this is done.  If someone gets their DNA "sequenced" at 23andMe, for example, they are not getting their whole genome sequenced.  They are getting individual bits believed to be possibly clinically relevant sequenced.  It's a different technology than you would use to get the whole genome.  A whole mammalian genome is really big, therefore kind of expensive to sequence, and is mostly non-coding DNA of questionable relevance.  Therefore you can just genotype the exact bases of interest, or maybe do exome capture, which gets you full sequence for the 1% or so of the genome that is currently annotated to be coding.  Obviously either of those approaches would no be all-inclusive, so one might wish to redo them at a later date with new targeted regions of interest.
Even if you did get a whole genome done with current technology, right now it has limitations, namely read length, that make it hard to work out certain details.  I could easily see someone needing to redo a genome using a longer read technology, when that technology arrives.
Also, we aren't really discovering that many new genes. Certainly not protein-coding ones.  What is more likely is that we discover that variants in this or that gene might be clinically relevant to some health parameter.

Apart from what WYSIWYG said.
It might also be the case that you need to re-sequence because you're interested in seeing the changes that might have occurred since the last sequencing. Remember that cells keep dividing and therefore changing, so the DNA is slightly different from one cell to another (and from one population of cells or tissues to another). Single-cell sequencing is almost here.
The obvious example here is a tumour. It helps knowing what happened and if possible detect the driver (or drivers) mutation (cancer cells tend to increase their mutation rate).


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How do I view the simulated protein structure along with ligand in VMD?
I did a simulation of a protein-ligand complex, and it has stabilized after 5 ns. I have got the respective pdb files for every ns, but when I try to view in VMD (Visual Molecular Dynamics), I cannot see the ligand. How do I select the heteroatom in the graphical representation window? My complex has 1129 + 50(ligand atoms) but while loading it just shows the protein molecule(1129).

->www.biostars.org

After you load the molecule in VMD, from the menu bar select Graphics->Representations... In the box labeled Selected Atoms, type "hetero" and hit enter. You should now see only your ligand (and whatever other non-protein/DNA atoms that happen to be in your PDB file). Hit the Create Rep button, enter "protein" in the Selected Atoms box and hit enter (you can see a list of all the possible selection words in the Selections tab). You will now have a separate representation for your protein and your heteroatoms, and you can modify them with the other settings in the Graphical Representations dialog in order to make them visually distinct from one another.
If this doesn't fix your problem, open up your PDB file in a text editor and check to make sure that there are lines that start with ATOM or HETATM that correspond to the atoms in your ligand. If they aren't there, your problem isn't with VMD but with one of your prior manipulations.


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Why is yawning contagious?
Why does yawning seems to be contagious?
Is this similar to laughter being contagious or does it has a different reason?

Are you looking for a causative factor (i.e, a neural circuit connects external yawing cues to the internal yawning effector center) or are you interested in the physiological/evolutionary reason (i.e., yawning communicates hypoxic environment to other individuals in the nearby).

I would be interested in the physiological/evolutionary reason.

Yawning is contagious in humans, which means that, in general, it is more likely that a person yawning after perceived (by sight, hearing, or both directions) yawning issued by another person. The frequency of infection varies throughout the day, with a peak in early morning and late evening.
A recent study conducted by Ivan Norscia and Elisabetta Palagi (University of Pisa) has provided the first evidence that the behavioral contagion of yawning is associated with the empathic bond between people. The study showed that the infection follows the same gradient of empathy: is greatest in close relatives (parents / children / grandchildren, brothers, stable couples), decreases in friends, then acquaintances in (people connected only by a third, external, ie work or a friend in common) and reaches a minimum in the unknown.
Various studies of clinical, psychological and neurobiological suggest and support the link between the yawn contagion and empathy. For example, the infection begins to occur at 4-5 years of age, when children develop the ability to identify, in a proper way, the emotions of others. In addition, the infection is reduced or absent in people who have problems related to empathy, how people with autism, and is positively related to subjective scores of empathy based on tests of psychological type. Finally, the areas of the brain related to the emotional sphere overlap, in part, with those involved in the response to yawn, with a possible involvement of mirror neurons.
Yawning has been observed among various primates. In these cases the yawn is a threat gesture, a way of maintaining order in the primates' social structure. Specific studies were conducted on chimpanzees and stumptail macaques. A group of these animals was shown a video of other conspecifics yawning; both species yawned as well. This helps to partly confirm a yawn's "contagiousness".
In animals, yawning can serve as a warning signal. For example, Charles Darwin, in his book The Expression of the Emotions in Man and Animals, mentioned that baboons yawn to threaten their enemies, possibly by displaying large canine teeth.
see yawn
autism
Gordon Gallup, who hypothesizes that yawning may be a means of keeping the brain cool, also hypothesizes that "contagious" yawning may be a survival instinct inherited from our evolutionary past. "During human evolutionary history, when we were subject to predation and attacks by other groups, if everybody yawns in response to seeing someone yawn the whole group becomes much more vigilant and much better at being able to detect danger.
Personally, I can make an observation. Cats yawn as specific means to calm people, and especially to reassure the other cats and the pups. In order, they stretch, yawn, lie down, half close theyr eyes as if they start to sleep. When the whole group repeats the gestures, the majority of them (now calm) actually falls asleep.
EDIT @kmm

Norscia & Palagi
The Pisa Group in cooperation with CNR Consiglio Nazionale delle Ricerche =
Friends and loved ones yawn together - Science Daily
The Pisa Group/ Elisabetta Palagi, Ivan Norscia, and Daniela Antonacci - University of Michigan - Dearborny
Gordon G. Gallup

Could you add some links to the studies you cite (Norscia & Palagi; Gallup) so that interested parties can follow up with the primary literature?


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What mechanism suppresses melatonin release in humans?
I'm looking at melanopsin, a photo pigment in the human eye. One of its actions is to trigger suprachiasmatic nuclei SCN to suppress melatonin release. I'm interested in learning what is the exact mechanism that actually suppresses melatonin release. Is it another hormone or some way of modulating the pineal gland function?

Is this not answered within your own previous question?
http://biology.stackexchange.com/questions/3614/how-are-melatonin-levels-measured-in-humans?rq=1
"melatonin release is suppressed by blue light with peak suppression occuring at 420-480nanometer range.
This light is registered by Melanopsin, a photopigment in the eye.
Melanopsin signals the Suprachiasmatic Nucleus, the master clock of the human body.
Suprachiasmatic nucleus makes a decision to suppress melatonin release in response to blue light observed."

So you're interested in the nature of the signal between melanopsin and the SCN?

To clarify: I know that SCN makes a decision to suppress melatonin. What interests me is what takes place during the actual process of suppression. What physically ensures that pineal gland stops producing melatonin. Is it another hormone that is released?

From Kalsbeek et al 1999 Neuroscience (link 1, link 2)

"retina-mediated photic activation of suprachiasmatic nucleus neurons
  induces the release of GABA from efferent suprachiasmatic nucleus
  nerve terminals, resulting in an inhibition of melatonin release by
  the pineal gland."

So it appears that GABA is key. When the retina is stimulated by light it results in activation of the neurons within the SCN which then release GABA. There is much more detail within the article and this is not my field so you may get more from reading that rather than my summary attempts.

Take a look at this (especially page 938): Coordination of circadian timing in mammals by Steven M. Reppert & David R. Weaver Nature 418, 935-941 (29 August 2002) | doi:10.1038/nature00965

Sodium-dependent action potentials provide the primary means by which
  the SCN transmit circadian outputs to other brain areas and are
  essential to its role as a pacemaker1.
Signalling molecules from SCN efferents include neurotransmit- ters
  and secreted factors. An output role for secreted factors comes from
  the results of SCN transplant studies that suggest that the
  alternating activity of SCN-derived ‘inhibitory’ and ‘activating’
  factors drives locomotor activity rhythms (and hence rest/activity and
  sleep/wake cycles) in rodents

There was also a mention of per/cry gene expression.

Welcome to Biology SE.  It would be helpful if you could summarize the findings in your own words: you cannot count on everyone having access to the journal, or the link even working in the future.

Sure, will do :-)

Thanks for a great article! It does not explicitly state how the pineal gland is affected though.


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Problems with analysis of small RNAseq data - Adapter trimming
I have always faced a problem while analyzing small RNAseq data, at the step of adapter trimming.
Overview of small RNAseq (Illumina)

RNA is size fractionated using columns or PAGE
3' and 5' adapter ligation
cDNA synthesis
PCR amplification
Sequencing

The length of the reads is dependent on the machine and recent ones like HiSeq can provide ~200bp.
The problem, however, is with reads smaller than the max read length of the machine and this is common with small RNAs such as miRNAs (especially if the concatemer of smallRNA and the complete 3'adapter is bigger than the max read length).
The first step of analysis is trimming of the 3' adapter (Illumina Truseq: TCGTATGCCGTCTTCTGCTTGT).
Several algorithms are available to do this job and what they precisely do is to check for overlaps between the adapter sequence and 3'end of the reads and then clip the aligned region.
Now the problem is this
You can't be really sure of very small alignments because they may not really be originating from the adapters, which means that you should specify a lower limit of alignment for clipping. I usually set it as 5 (intuitively).
But if a small piece of sequence really came from the adapter then it will remain and there is no way to clip it without any doubt.
The real problem comes during aligning the reads to the reference sequence. Aligners like bowtie (which i prefer to use), generally have user defined argument for number of allowed mismatches. Bowtie generally doesn't perform very well if you allow a lots of mismatches.
Subsequently, you might lose a really valuable read.
Alternative
To avoid this problem I sometimes trim the reads to around 25nt (for miRNA profiling). This creates a new problem:
You can't really distinguish whether the read came from a pre-miRNA (a longer RNA) or from mature-miRNA (smaller RNA that arises by processing of pre-miRNA)
Does anyone have an experience or an idea about how to solve this problem?

I would first recommend you ask this question biostar as the subject matter you are inquiring about is much more relevant there.
That having been said, you have another option which is to use an aligner that soft clips 3' ends of reads specifically to account for adapter (or polyA, or whatever) contamination that might have flew under your radar.
STAR is such an aligner. If you search their mailing list archives for "clip", you should find several posts related to your question.

Thanks Steve.. But I wonder why this forum is less suitable.. my purpose of joining here was to have all biology in a single forum without having to have multiple accounts.. i have an account in seqanswers and i shall ask this question there. but i am slightly disappointed by this concept of separate niches (especially when it all comes under same area of study)..

@WYSIWYG: I suggested biostar, not seqanswers ;-) Still, this forum is less suitable simply because your question is more of a bioinformatics nature than strictly biology one -- and you will find more expertise related to bioinformatics on biostar than here. I would imagine that there are likely many people on biostar who are strong bioinformaticians that do not have accounts on this site, and you would benefit from getting their input. Consider, for example, why there are different SE sites for math and stats :-)


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Iron deficiency anemia symptoms explanation
In my classes and on the internet, brittle nails and dry hair (or hair loss) are described as symptoms of iron deficiency anemia, but none explains the cause of such symptoms.
Does anyone have any clues or explanation of such symptoms?

http://en.wikipedia.org/wiki/Iron_deficiency_anemia#Signs_and_symptoms

Can you provide the link to the sources that say so?  Because frankly I havent heard of these as anemia symptoms

It would likely depend on the type of anaemia - deficiencies in iron, B12 & folate, anaemia of chronic disease, haemolytic anaemia etc.  I haven't heard of this as a symptom of anaemia myself, however it is possible that it could be as a side effect of the problem that is causing anaemia (anaemia never being a spontaneous disease, always having an underlying cause).

Another clarification I would like to make. Anemia is a condition which is defined by reduced RBC count. Anemia is one of the things that Iron deficiency causes. Other effects of iron deficiency can be syndromic or even correlated (without dependence) with anemia but cannot be called as symptoms of anemia.
And as @RoryM mentioned there can be other reasons for anemia.

The molecular basis of copper-transport diseases in Trends in Molecular Medicine, Volume 7, Issue 2, 1 February 2001, Pages 64–69, has a link to a 1973 paper by JM Gillespie entitled "Keratin Structure and Changes with Copper Deficiency," stating

Menkes patients are often diagnosed from their unusual hair structure – termed pili torti – also known as kinky or steely hair, caused by reduced keratin cross-linking, a process that is catalyzed by an uncharacterized copper oxidase.

I haven't been able to find anything further, but since iron and copper levels can both be affected in anemia, this may be the link you're looking for. There may also be other enzymes involved that use iron-mediated catalysis. Since there are so many different kinds of keratins (the major protein that makes up hair and nails, among diverse other functions in the body), there may easily be multiple cross-linking and assembly pathways.
Other websites I found have also claimed that anemia-associated pathologies with hair and/or nails may be related to reduced oxygenation as a result of low iron levels, but they don't give any citations, and given the nature of the sites (nail care, fashion, etc.) it may just be an educated guess.

"but since iron and copper levels can both be affected by anemia" ..... It is the other way round. Iron deficiency causes anemia not vice versa. A small addition to your answer (some guesswork): proline and lysine hydoxylase require iron cofactor and this enzyme helps in forming mature collagen. Nonetheless the symptoms mentioned in the question are unlikely to be first signs of iron deficiency

sorry, I could have worded it better, I was in a hurry :) Yes, anemia (in general) is defined as a deficiency in something, and in common usage the word anemia alone is typically taken to mean iron anemia or iron deficiency. What I meant is that copper levels can also be affected by iron anemia.

But do you think collagen synthesis can be affected ??

@WYSIWYG I'm not sure what collagen has to do with it - fingernails and hair are make up primarily of keratins...

it has got nothing to do with fingernails but the thought just popped in my head that iron deficiency may affect ECM. NOnetheless, all this fingernail and hair symptoms seem dubious to me.. I cant find any reliable source which claims so

I did some searches on the relationship between iron deficiency anemia (IDA) and hair loss. Surprising, there are lots of debates about this and I don't think anyone has a solid reason.
If iron deficiency is related to hair loss then taking iron supplement may potentially reverse the effect. However, this article pointed out that:
....there is insufficient evidence to recommend giving iron supplementation therapy to patients with hair loss and iron deficiency....
My understanding is, human body actually contains very little amount of iron. And most of the iron are involved in cellular respiration and oxygen exchange. Free floating iron is very toxic and bad for the body. Therefore most of the iron storage are in Hemoglobin (2/3), followed by ferritin+hemosiderin (about 1/3), some are utilized by the mitochondria (those awesome cytochromes and electron transport chains), lastly the transport vessel trans-ferritin.
I think Hair loss and brittle nail are just indirectly caused by IDA. Because most of the time IDA is cause by malnutrition. And usually the food sources that are contained the most iron are meat, poultry and egg. These food are also rich in protein. I think lack of meat protein-rich diet is the main reason of hair loss and brittle nail, and such diet leads to IDA. (Strict vegans, be careful about your food choices!)

There are several non-animal sources of iron- spinach, beans etc.

@WYSIWYG You are right. But as I said above, I think hair loss and brittle nails are not directly caused by IDA. It's caused by malnutrition.

Roger that.. :) [somehow the name tagging is not working]


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Is there any relation between weight class and extreme lifting performance in O-lifting?
We usually thought that weightlifting is a tough game. It is only for great physique.
There are 6 man in history who lifted  3 times their body weight. Check here.
3 of them are Bantamweight(56kg), 2 of them are Featherweight(60kg), and 1 is at Lightweight(67.5kg).
All of them are light. Is there are fact that light weighted are more efficient in weight lifting?

I suspect that such might partially result from both an inherent advantage and a coincidental advantage of lower height.  The former would be an effect of strength being roughly proportional to the cross-sectional area of muscle (square of a linear measure) and mass being proportional to the body volume (cube of linear measure).  This would make a 182cm person 10% stronger by body weight than a 200cm person of the same build (when one might initially think that having the same build would imply the same strength per unit weight).
The second advantage might be from taller individuals tending to have a more slender frame.  A more slender frame might be associated with lower muscle bulk (cross-sectional area) even with intensive training (more "ectomorphic"?).  Since there is a limit to thickness, to increase mass height must be increased, and if taller individuals tend to be more slender in build then as mass increases the build would tend to become more slender reducing muscle cross-sectional area (strength) in proportion to mass.


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Where to put the gene after eukaryotic promoter for best expression levels?
As far as I know there is an optimum distance between a promoter and the gene for the best expression levels. What is that distance for common promoters like CMV, SV40? If you have a first hand experience about this (e.g. if you have cloned the gene way downstream of the promoter and didn't/did see expression) and share it I will be grateful.
If you have comprehensive knowledge for other promoters please feel free to add them in the list too; they don't need to be common. I just want to hear people's experience on the subject.

a 10bp linker is sufficient. From all your other posts I gather that you are trying to build a synthetic system. My personal strategy for things like these is to pick up sequences from the vectors in your lab that you know work quite well.

Eukaryote is rather general - different eukaryotes have vastly different regulatory systems. Are you talking about something like human or something like yeast?

Take a look at the sequences for some common/popular vectors for your target organism. As WYSIWYG pointed out, it doesn't need to be very big, and it can vary somewhat depending on where in the multiple cloning site you insert your gene of interest.

Thank you all for your comments. I did look to other common constructs and decided to leave around 40 bp between promoter and the initiation codon (this was the minimum distance I could leave because of the restriction site limitation). @Bitwise, I am using mammalian cells (specifically; fibroblast and MDCK cells).

I used pEYFP-N1 (stock vector) where the spacing between the CMV promoter and the EYFP start codon is ~88 nt, and the expression is high.
I also cloned a protein 5' of the EYFP, only 4 nt after the CMV promoter. The expression of this fusion was also high. Because I was only using the chimeric protein in experiments, I did not try to assess differences in EYFP expression.
If you look here: https://synbiota.com/projects/19/sequences you'll see the basic pEYFP-N1 vector and the separation between the CMV promoter and the EYFP coding sequence.
On wiki: "The TATA element and BRE typically are located close to the transcriptional start site (typically within 30 to 40 base pairs)."


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What is the translation termination efficiency in mammalian cells?
When I express proteins in bacteria I put at least two stop codons at the end of the gene to increase the termination efficiency. Is this the case in eukaryotic cells too? If I put a single stop codon is there a risk for the ribosomal complex to readthrough and continue translation?

There is no such practice as far as I know. Personally I have never done that and things work fine. You can add a polyadenylation signal, though.
Is this a synthetic gene or something ?

From what I understand the eRFs are not perfect but they still work better than RF1 and RF2 which allow for a non trivial amount of readthrough.

This is a synthetic gene. I already designed a primer with 3 stop codons at the end of the gene. I will post here again if I see any improvement in gene expression (with compared to other similar constructs in our lab).

There is a paper for transfection of mammalian cells here which has a bit of comparison of stop codon and protein yield.
Otherwise this article suggests having a 'rare' codon after the stop to prevent readthrough. But yes, I'm not sure why one wouldn't simply put two stops after each other.


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What biochemical molecule viewer allows for changes in amino acids and resulting tertiary structure?
I am familiar with the Jmol, Rasmol and PyMoL softwares, and was recently introduced to BioBlender. However, I am completely unaware if any of these programs (or others) are capable of loading a .pdb and allowing for the change in a particular amino acid, to ultimately visualize the change in tertiary structure caused by that substitution.
Are any programs capable of performing an amino acid substitution on a molecule loaded by .pdb and showing the change in tertiary structure?

Pymol should have that capability... try Gromacs

This may help: http://www.pymolwiki.org/index.php/Mutagenesis

One application that I am aware of that will do this, as well as much else besides, is Swiss-PDB Viewer aka DeepView. If you go to the site and select User Guide in the left hand tabs you will see a subsection called mutations and following that will tell you what the application can do.

Visualising the change induced by an amino acid on the 3D structure is part of an entire field of research call molecular modelling (http://en.wikipedia.org/wiki/Molecular_modelling). There is a huge body of research available on the subject and I am not up-to-date anymore in this field.
What I used to do was simulating the optimal shape of a AA chain (~10) which was part of the bigger molecule. The technique consisted in using a Monte-Carlo procedure to simulate heating the molecule and cooling it down millions of time and checking for the stability of the resulting conformation using . I used Modeller, Procheck and Prosa. That was in 2000.
There is a wikipedia list of molecular mechanics modelling software here (http://en.wikipedia.org/wiki/List_of_software_for_molecular_mechanics_modeling)

If I understand what you're attempting to accomplish, as David said, this is a vigorous area of study in structural biology. I'll start by saying that a single amino acid substitution will often make virtually no difference in a structure, but there are proteins where a couple substitutions will convert an all alpha-helical protein to all beta-sheet, so I'll leave it to you to judge what's worth trying. There are three routes you can take:
First, for small changes, pymol does have a built in force field ("molecular sculpting") and residue mutagenisis.  Chimera has a tool called "minimize structure" that are probably better developed. And VMD has the force field tool kit plugin.
If you expect larger changes, you might go with a full simulation package like NAMD or CHARMM.  They would take significant effort to get running, but they're there.
Finally, you can just go with an empirical structure prediction server. All you do is open your sequence file, make your substituions there, upload it to the server, and wait for them to email you the results.  They'll do sequence alignments to find relevant PDB files and everything. I recommend the Robetta server (running David Baker's Rosetta). http://robetta.bakerlab.org/


text:
What causes peanut allergies?
Surveys show that 0.6 percent of the US population has an allergic reaction to peanuts. This prevalent allergy has even prompted some to ban peanuts from schools altogether.
I am completely unfamiliar with the topic, and can't seem to find any specifics in the literature. Thus my question is:
What specifically causes the the allergic reaction associated with peanut consumption, and why is it so prevalent in comparison to other foods?

This article claims that a protein complex Ara H has the allergenic properties.

I just wanted to let you know that I changed the title to reflect that the question is about the causative agent of allergies, rather then the epidemiology of allergies.

Ara H 1, Ara H 2, and Ara H 3 are the three main peanut allergies. AH1 and AH2 are typically higher-acuity anaphylactic type reactions. The molecular cause for this is that some Immunoglobulin E develop a serum sensitivity and bind directly to vicilin and conglutin (the active proteins in AH1 and AH2). There are actually 7 or 8 Ara H complexes, but it is believed the remaining ones have a lesser affinity for IgE, reducing the frequency of allergic reactions and thus their availability for relevant studies.
The prevalence of the allergy is generally attributed to a combination of exposure and genetics. Many people have a genetic predisposition to certain allergies, but to a certain extent the body must be exposed to the allergen before an allergy will develop. The high use of peanuts in the food industry, and even to a lesser degree in skin care products like lotions, can account for early exposure resulting in allergies.
From the other direction, some research suggests that early and frequent exposure from infancy through childhood (albeit the choking risk) can reduce the demographic prevalence of an allergy in a given population.


text:
PTMs of proteins via mass spec?
I understand that mass spec is widely used to study PTMs like glycosylation of proteins, but how can mass spec determine correct PTM structure of say glycosylation if two glycan structures have the same mass but different arrangement (i.e. isobaric)? Also, how can mass spec determine the difference between structural isomers?

Based on fragmentation patterns.

@WYSIWYG: could you elaborate (in an answer)?

I am not a MassSpec expert but this is what I understand from my spectroscopy basics:
In tandem MS (MS-MS), the ionized analyte generally undergoes or made to undergo fragmentation (breaking up into smaller ions). Fragmentation allows you to study finer details. It is also essential for de-novo peptide sequencing. Different fragmentation techniques, such as Collision Induced Dissociation (CID) or Photofragmentation, yield different kinds of secondary ions.
You can intuitively understand that Fragmentation of structural isomers will yield different kinds of secondary ions (using the same fragmentation technique). The choice of fragmentation technique depends on the molecular nature of the analyte. In the article that I mentioned previously, they use laser induced photofragmentation to dissociate glycan-ions.
Even small molecules, which had been classically studied by MassSpec, undergo isomerization after ionization in order to stabilize the ion. Reactions such as reverse-Diels-Adler can also happen. The pattern of reactions would differ between structural isomers. For basics you can refer to a book on Spectroscopic techniques by Silverstein


text:
What is the cause of muscle cramps?
According to Wikipedia, muscle cramps are caused by the inability of myosin fibers to break free from the actin filaments during contraction, resulting in a prolonged contraction.
A lack of ATP would obviously produce cramping, as myosin requires ATP to become free from actin.
However, I have heard that potassium and sodium are useful for treating cramps and that their deficiencies can lead to cramping.
So, how does a deficiency in sodium or potassium result in the inability of myosin fibers to break free from the actin filaments during contraction?

check this article. It is not accessible to me.

The quick and simple answer:
Cramps of a hypokalemic origin are much more common than those of a hyponatremic origin because the Na-K pump is more effective at moving potassium in comparison to sodium.
At the onset of a muscle contraction, the presence of calcium triggers the opening of the Na-K channels in the membrane. Potassium is a calcium inhibitor, so as the potassium flows out of the cell, it eventually reduces the presence of calcium. This causes the closure of the Na-K channel (negative feedback mechanism). In a hypokalemic state, the lack of sufficient K doesn't inhibit the calcium channel, and in turn doesn't properly terminate the muscle contraction at the cellular level.
The continued presence of calcium, which has a lot to do with nerve impulses, means that the nerves keep firing, and in some cases such as a 'charlie horse', these impulses fire fast and continuously. The body at this point is reaching a small local state of metabolic acidosis resulting from the extremely high oxygen consumption, increasing levels of CO₂ (the acidosis component) and reducing blood partial oxygen levels. Since the oxygen is no longer as abundant as it was, it inhibits the bodies ability to locally produce the ribose and phosphate necessary for ATP. Less ATP = more myosin that can't be disconnected from actin = continued muscle cramp.

still doesn't answer why low sodium causes cramps..
the exercise cramps dont happen because of hypokalemia.. potassium is hardly lost in sweat.. Potassium is not a calcium inhibitor.. calcium release in muscles in voltage gated, which is triggered by sodium influx

"Muscle cramps, notably nocturnal leg cramps, are common symptoms experienced by general medical patients, particularly the elderly. Their etiology is varied; most commonly, these cramps are idiopathic. With the notable (and relatively rare) exceptions of serious electrolyte imbalances including hypokalemia, hypomagnesemia and hypocalcemia, these cramps are typically benign in nature." So electrolyte imbalances are not a common cause of cramps, even in the elderly.

And (same source): "The second recent study to challenge the notion that diuretics are a common cause of muscle cramps was a cross-sectional survey of 365 general medical patients. Although the prevalence of leg cramps in this cohort was 50%, no associations were found between these cramps and any medication except analgesics that were used to treat the cramps." (Most diuretics lead to a loss of potassium through urine; there are some potassium-sparing diuretics but they were not distinguished in this study.)

Furthermore the [review] paper says "Interestingly, potassium‐sparing diuretics, such as amiloride, are also associated with cramping."

And a more recent (2012) investigation: "Physicians may be surprised that potassium-sparing diuretics have a stronger link to cramp treatment than loop diuretics. Yet, hyperkalemia facilitates neuronal excitation, and hypokalemia suppresses motor neuron activity." So I'm having doubts that your claim that cramps of a hypokalemic origin are much more common [...] has a basis in epidemiology.

Muscle contraction occurs when the brain tells the body to move. The brain then starts an action potential down the motor neurons, until it reaches the terminal bouton. At the terminal bouton, it releases the neurotransmitter, acetylcholine, which travels through the myoneural junction and into the myoneural cleft. ACH binds to the receptor, which causes an action potential in both directions along the cell membrane.
    The action potential repels the potassium, which travels down the cell membrane until it falls into a transverse tubule. K+ continues to fall into a transverse tubule and accumulates, which increases voltage (-70 to -50 mV). The voltage change causes the calcium gates to open and to diffuse the calcium.
    Calcium then binds to troponin, which in turn binds to tropomyosin and pulls it, exposing the g-actin binding site that allows myosin and actin to bind. The myosin head changes shape (called power or working stroke) and pulls the actin towards the M line, and the muscle contracts. Similar to a tug of war, the myosin heads (your hands) pull on the actin (the rope) to contract a muscle. Like team members in a tug of war, the myosin heads alternate between pulling and holding on to the actin; the only way that myosin will release actin is to add ATP, which forces the two apart and thus relaxes the muscle (as mentioned above).
Now, a lack of potassium, sodium, or calcium would prevent the muscle from contracting but won't relieve a muscle cramp. The common advice of eating a banana actually does help relieve cramps but not because of the potassium. Bananas also have sugar and fat, which are converted into ATP.
Muscle cramps are primarily caused by a lack of ATP in the body. ATP forces the myosin to release the actin; thus, the muscle relaxes and the cramp is relieved.
Another cause could be the lack of magnesium, which helps the ATPase sodium/potassium pump, which, in turn, returns the voltage to resting potential and relaxes the muscle.

Anaid, welcome to Bio SE, and thank you for resurrecting this question with an answer. If you can try to add in line citations as mentioned in the help section.  I also think there are some more topics that might should be covered here like Ca2+-ATPase.

None of these answers makes sense, and there some minor errors in them. I know that action potentials may continue to fire down the axon. One reason may be because acetylcholinesterase is not coming to the rescue and cleaning up all the acetylcholine in the synaptic cleft. But that usually happens if there are drugs involved. So, in terms of natural muscle cramps, it really does not make any sense whatsoever.
A really good reason is mainly because of a lack of potassium. The function of potassium inside the muscle cell is to repolarize the membrane. However, if there is not enough potassium, the time it takes for it to repolarize is very slow. On the other hand, sodium is used to depolarize the membrane. As a result, since there is an unequal distribution of sodium to potassium, less potassium flows out the membrane while more sodium flows into the membrane, causing it to depolarize faster than it repolarizes. Since the inside of the cell is becoming more positive with the help of sodium, and since the outside of the cell is becoming negative with the help of potassium, the inside of the cell is pulling back the potassium. But, again, there is an unequal distribution of potassium compared to sodium, so it will never achieve that electrochemical equilubrium (resting membrane potential). Since electrochemical equilibrium can not be achieved, sodium is flowing inside the membrane, causing it to depolarize across the membrane and to form an unfused tentanus at a rapid rate.
The rate in which this is happening is so fast that it won't let the myosin head detach from actin for a long period, so it is continuously making the sacromere shorter, causing a pain receptor to travel toward the CNS. This is my hypothesis.

Can you please expand your answer with some references?

One possible reason why low sodium levels induce cramps may lie in the cation selectivity of the nicotinic acetylcholine receptor (nAChR), which is an ion channel. When ACh binds to the receptor site on the nAChR protein, the protein changes shape to open a pore formed by the protein in the cell membrane. This pore allows the influx of both sodium and calcium ions, which induce depolarisation (a positive shift in the membrane voltage) to trigger the voltage-dependent ion channels responsible for action potentials.
A reduction in sodium ions would mean more calcium is able to enter the cell through the nAChR. The calcium ion carries greater charge as it is formed by the loss of two electrons from its outer shell, while the sodium ion is formed by the loss of one electron. This means that, each time the nAChR channel opens, the cell undergoes greater depolarisation. The muscle response is harder and faster to the same neural stimulus, which results in a depletion of energy stores.

One of the things that I would like to point out is that ATP detached the myosin head but does not control the powerstroke! If there are no more ATP being produce, then rigor mortis results. You will just be really tense/sore because the myosin head is not being released, and it's staying contracted (perhaps a fused tentanus), but it does not make the sacromere shorter if ATP are no longer able to be produced. And if there are no more ATP being produce, it can't be hydrolyzed into ADP and inorganic phosphate (Pi).
That would help to create the cross bridge. In addition, inorganic phosphate, not ATP, is the essential compound when you want the power stroke to happen.

A quote from Flex Pharma:

Most muscle cramps are not caused by dehydration, lactic acid,
  electrolyte imbalance, or muscle tightness. That is why popular
  remedies like sports drinks, bananas, magnesium tablets, and
  stretching are usually ineffective. New research has shown that cramps
  and spasms do not originate in the muscle itself, but are caused
  instead by a neural mechanism: excessive firing of the motor neurons
  in the spinal cord that control muscle contraction.

Activation of transient receptor potential (TRP; TRPV1 and TRPA1) ion channels reduces muscle cramps.

http://www.flex-pharma.com/scientific-presentations.php


text:
How the concentration of an antibiotic is chosen for YPD medium?
I need to prepare YPD plates containing hygromycin, but I don't know which concentration I can use. I'll select transformants of Hansenula polymorpha.

Presumably this will depend upon the promoter that is driving expression of the marker, and the copy number of the plasmid. Do you know anything about either of these parameters?

First I'll linearize this plasmid. because I want it to be integrated into the genome. (linearized DNA integrates to the genome in H. polymorpha). So there will be no additional copies. And tef1 promoter is present before hygromycin gene.

According to this site,
for yeast (I assume this means Saccharomyces cerevisiae) you should use 50 µg/ml to 200 µg/ml; for fungi (!) use 100 µg/ml to 300 µg/ml. The site also stresses the importance of the pH of the medium.
I think that, unless a Hansenula expert comes along,  you will have to try an initial experiment to measure the sensitivity of untransformed cells across the range indicated. Then you can try selecting resistant cells at a concentration somewhat higher than that which kills untransformed cells. Finally, you can check to see how resistant the transformants are, to enable you to decide on a useful concentration for routine selection of transformants.


text:
How do omega-3 fatty acids reduce cholesterol at the molecular level?
I have heard that omega-3 fatty acids found in fish are a good way to prevent/reduce cholesterol problems.
My question is how do omega-3 fatty acids do that: what are they doing on a chemical level to produce this effect? And is there any way to maximize this effect through other substances, that may work in concert with them?

What do you mean when you say "maximize this effect through other substances"?  Are you asking if there are co-factors that have no/little effect on their own, but act synergistically with omega-3 fatty acids to reduce cholesterol problems? Or are you simply asking if there are other substances that reduce cholesterol problems?

The former. Is there anything that works with them.

i dont think they REDUCE cholesterol.. omega-3 fatty acids comprise the essential fatty acids (omega 3 refers to the position of double bond from non carboxyl end).. and BTW cholesterol is also important for the body- it makes up the membranes and also serves as a precursor of vitamin D [sunlight basically converts 7-dehydrocholesterol to vit-d]. excess causes problems

the above answer is going in the right direction - unsaturated fatty acids keep the membrane more pliable and cholesterol, which is an inflexible planar molecule tends to make it more rigid I think.

@shigeta - Re cholesterol"... cholesterol possesses a slick and rigid structure. ...this would naturally result in a stiffer membrane. Yet, for the past 10 or so years, physicists and biologists have assumed that cholesterol had nearly no effect on the stiffness of membranes ...", https://www.sciencedaily.com

@PeterBernhard its funny i heard even in grad school that they made the membrane stiffer.  in any case as they are linked to hypertension I assume we are on the right track here - interesting factlet :)

I found this research paper (pdf) by D. Roger Illingworth, William S. Harris, and William E. Connor (Journal of the American Heart Association, 1984) that offers some help. Its abstract says:

Diets rich in omega-3 fatty acids derived from fish oils lower the plasma concentrations
of low density lipoproteins (LDL) and very low density lipoproteins in humans. The present study was designed to examine the mechanism(s) by which diets enriched in omega-3 fatty acids reduce plasma LDL cholesterol levels in normal subjects.

In the Dicussion part, you will find your answer:

Reductions in the plasma concentrations of LDL in human subjects consuming diets rich in long-chain omega-3 fatty acids from fish oils or omega-6 fatty acids from vegetable oils may be due to a reduction in LDL synthesis, an increased fractional rate of catabolism of LDL, or a combination of both... The present study indicates that the hypocholesterolemic effects of long-chain omega-3 fatty acids present in fish oils results from a reduction in the rate of LDL apoprotein B synthesis and that such oils do not stimulate the fractional rate of catabolism of LDL. These observations imply that the incorporation of omega-3 fatty acids into cellular or lipoprotein lipids does not enhance the rate of receptor-mediated catabolism of LDL as has been observed with omega-6 fatty acids in vitro.

I could not find a paper on its exact mechanism, so I'd rather say that the process is still not clear. Yet, as the paper suggests, the main effects of omega-3 fatty acids are reducing LDL synthesis and/or increasing fractional rate of catabolism (FRC) of LDL.
A similar article can be found here:

Omega-3 fatty acids have been shown to lower triglycerides, which are a type of fat in the bloodstream. Experts aren't sure of the exact mechanism. Omega-3 fatty acids may also slow down the growth of plaques in the arteries and reduce inflammation throughout the body.
A number of studies going back years have shown the benefits of fatty fish. In an important review of studies, researchers found that getting daily omega-3 fatty acids from fish oil could lower triglyceride levels by 25%-30%. The results were published in The American Journal of Clinical Nutrition in 1997.

BUT, I would also like to point out that the question itself "omega-3 fatty acids found in fish are a good way to prevent/reduce cholesterol problems" seems to be a matter of debate because of articles like this:

New data presented this week provide clues as to why eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)--both components of omega-3 fatty acids--have differential effects on LDL cholesterol.
Presenting the results of the laboratory study here at the National Lipid Association (NLA) 2011 Scientific Sessions, senior investigator Dr Preston Mason (Brigham and Women's Hospital, Boston, MA) said that EPA is an inhibitor of lipid oxidation at normal and elevated cholesterol levels in the presence and absence of DHA, while DHA seems to have no real effect on lipid peroxidation. This trial was one of a number of studies that attempted to address the clinical question as to why LDL-cholesterol levels increase in patients treated with the triglyceride-lowering omega-3 fatty acids. In his trial, Mason et al compared the effects of EPA and DHA--alone or in combination with statins--on lipid peroxidation in polyunsaturated fatty-acid– and cholesterol-enriched vesicles.

Re "may be due to a reduction in LDL synthesis,"
Do you have any more reference on mechanism which you state as not established yet?
Are there any "shunts" in liver, i.e. liver packing its "self-made", i.e. unsaturated fatty acids made from glucose,   into LDL,  in contrast to unsaturated fatty acids that it does not produce? Would you be interested in some other answer elaborating on that point?


text:
Is episome a plasmid or a virus?
A plasmid is a small DNA molecule that is physically separate from, and can replicate independently of, chromosomal DNA within a cell.
In general, in eukaryotes, episomes are closed circular DNA molecules that are replicated in the nucleus. Viruses are the most common examples of this, such as herpesviruses, adenoviruses, and polyomaviruses. Episomes in eukaryotes behave similarly to plasmids in prokaryotes in that the DNA is stably maintained and replicated with the host cell.
Source is wikipedia.

A DNA molecule that replicates independently of chromosomal DNA is an episome. By this definition a plasmid is (usually) an episome. If a plasmid integrates into a chromosome by some mechanism (as for example in Hfr strains of E coli where the F plasmid is integrated) the plasmid loses its episomal status. Is this what you are asking?

this was exactly what I asked. Thank you very much.

(Reposting my comment as an answer since it seems to be what was required.)
A DNA molecule that replicates independently of chromosomal DNA is an episome. By this definition a plasmid is (usually) an episome. If a plasmid integrates into a chromosome by some mechanism (as for example in Hfr strains of E. coli where the F plasmid is integrated) the plasmid loses its episomal status.
The term "episome" was first coined by Joshua Lederberg to describe the nature of the F element in E. coli.

I'll have to disagree with Alan. If the plasmid is integrated into a chromosome it doesn't lose its episomal status. Actually the integration of the plasmid is a characteristic of an episome.
This is a quote from this article: http://www.qub.ac.uk/mlpage/courses/level3/plasmidhistoryreview.pdf

"... For a decade or more it was confused with ‘‘episome,’’ although that was carefully
  crafted (F. Jacob and E. L. Wollman, 1958, C. R. Acad. Sci. 247, 154–156) to mean agents with
  traffic in and out of chromosomes.

Also this book (https://books.google.pt/books?id=YnXB54RtrR8C&pg=PA132&dq=episome&hl=pt-PT&sa=X&ei=oKZgVd3HEZOu7AaeoYPABQ&ved=0CCoQ6AEwAg#v=snippet&q=episome&f=false) clearly mentions that an episome is a genetic element able to exist in an integrated form (associated with the chromosome) but also with it's  own origin of replication (autonomous form). So in fact an episome can be a plasmid or a viral genome, but not all genetic elements have this ability.


text:
How long can you keep sheep eyes in formaldehyde before dissecting?
We're preparing for an experiment in our classroom and my teaching partner and I are at different points in the unit.  I am concerned that the experiment will be less successful because of the freshness of the eyes.  How long can they eyes be stored in the fridge before they have to be dissected and is freezing them an option?

Formaldehyde is a preservative, and is used to prevent decay. As long as the tissue is completely perfused (and there shouldn't be any issues with eyes that I'm aware of) it can be kept essentially forever. Have fun!

you can keep the samples in 4 degrees. Sometimes, in certain tissues, formaldehyde preservation makes them brittle. Or sometimes it makes certain slimy tissues easy to dissect. Nonetheless, it wont damage the morphology. And if you are not interested in protein/RNA isolation, then you can store the tissue as long as you want.