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Why do I get such a strong background in a detection of DIG-labeled DNA (Southern Blot)?
I have done a Southern Blot Analysis of DNAmt transferred to nylon membranes. The DNA was firstly loaded on a 2% agarose gel. An immunoassay was done to detect the bands using Anti-Digoxigenin antibody with Alkaline Phosphatase which catalyzes a colouring reaction with NBT and BCIP.
The results has a very strong background as you can see in the following pic:

I am not sure about what can be the reason of that background. I have read that thick agarose gels produce strong backgrounds, but I never experienced that before. If somebody has experience on this or alternative explanations, I would thank him/her to share them with me.

Could you please describe your procedure? It is extremely difficult to pick apart what may be wrong here when we have no idea how you made the gel, how long you placed the anti-body on, what you blocked with, etc etc.
2% is not a terribly thick gel. It's higher than the norm (~1%) but I've never had this trouble with them. My guess is you did something weird when placing the antibody or blocking. Perhaps made the buffer improperly.

I agree, try to post the full protocol step-by-step (e.g. 1, 2, 3) since sensitivity can be altered by a number of mishaps that can occur at different steps of the process!

Thanks FrankyG and Kendall. I will write a short traslated version of the protocol and will link it here in a while. It is the first time I follow it but I know it worked fine for other colleagues. Probably I did something wrong and am trying to figure out what. When I tell a thick gel I mean the mm of thicknes. I have read that "The recommended thickness for agarose gel is 3–4 mm; a gel thicker than 5mm will result in fuzzy bands and higher staining background." Source: https://www.thermofisher.com/us/en/home/life-science/pcr/elevate-pcr-research/agarose-content-with-tips-and-tricks.html

The protocol http://bit.ly/1QE3y7z

Offhand, I'd look at the blocking and washing steps (perhaps try a different source of nonfat dry milk). Be careful about contamination (alkaline phosphatase is ubiquitous). Also, you may be over-exposing - reducing reaction time might produce cleaner results. At the extreme end, you might try using peroxidase-linked rather than phophatase-linked antibodies.

I appreciate your suggestions R.M. It makes sense to reduce the reaction time. Over exposure could be the cause. I left it overnight. Next time I will check every couple of hours to find out if we can have a cleaner signal controlling this step. I will try also with another nonfat drymilk. I used a new package and brand, different of this used by colleagues in previous experiments. If nothing works perxidase-linked antibodies can also be a good alternative. Thanks a lot.


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2nd messenger mechanisms
Many hormones use 2nd messenger mechanisms, such as cAMP and cGMP, and also act on the same cells. For example calcitonin and PTH both use cAMP and have opposite effects.
How is it possible that the same second messenger molecule (cAMP) can have opposite effects on the same cells? Why are there more hormones compared to 2nd messengers - doesn't that reduce the uniqueness of each hormone? Shouldn't that make the function of any hormone using e.g. cAMP be the same as that of any other hormone using the same 2nd messenger on the same cell?

Yes. Got it! Thank you!

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Will climate change affect flower population?
There is a flower population elevated 1000 meters above sea level.  If climate change causes a 12⁰C increase in temperature over the next 10 years, what will happen to the flowers?

Its really hard to say for any given field of flowers and its hard to separate temperature from so many other factors that are accompanying climate change.
For example, rainfall is changing as well as CO₂ levels.  The pH of the rain may change because CO₂ becomes a mild acid when its absorbed into water. The speed at which the seasonal temperatures vary and how cold is the minimum temperature and how hot is the hottest day will cause parasites and infections which kill plants to appear.
To be sure the heat is important, but the timing maybe just as important.  The temperature is causing earlier blooms and later growing seasons.
Each different species is responding differently, but odds are that nearly any given field of flowers is going to feel it.  The temperature is important, but its sometimes hard to guess if its the specific culprit in changes.

Making one change in an ecological system is much like eating one salted peanut.
A:  If you only assume a 12⁰ change (which is really large) then the net effect is not only warmer but dryer. Higher temperatures mean faster evaporation, faster transpiration.
B:  Warmer may also mean that some pest has a limiting factor removed. Look at the problem northern British Columbia has had with Mountain Pine Beatle. The pines are fine with the warmer winters.  But the lack of cold winters has allowed the beatle population to explode.
C:  Some plants are adapted to a large temperature range. Look at the range for dandelion. So warmer temps may make them grow faster and set seed sooner.
D:  Some plants are adapted to a narrow temp range, and will die out.
One of the questions is, "If the change is slow enough, won't they just move up the mountain. In some cases yes.  We can look at turn of the century (1900) photographs of mountains, and we can see the tree line -- both at the top, where it meets tundra, and at the bottom where it meets prairie -- has moved up the mountain.
However it also depends on the soil. And soils neither move fast, nor are created fast.
Example:  Lot of articles think Canada will win big with global warming, because more northerly land will be suitable for farming. Not so. Most of the land that has reasonable soil is already farmed. The soil north of that point tends to be mostly peat moss bogs, and glacial till. In addition over half that country is the Precambrian shield -- endless miles of granite under 2-3 inches of moss and gravel, with bogs and lakes between. The equivalent of a rural gravel road costs millions of dollars a mile to punch through this country.

I don't understand the 1 peanut analogy.

No one eats one peanut.  They eat handfulls.  Similarly making one change in one parameter has a ripple effect through the entire system.

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What are examples of genes succesfully reproducing in ways that is in conflict with helping the individual creating offspring
Most of the time, our genes have common interests with our "self".
We reproduce well if and only if our genes reproduce well.
In some cases, like in bees, the haploid bees can survive better if they help their queen reproduce.
In humans and other species, are there samples that give individuals "incentive" to help non family member to reproduce for the sake of their genes.
So

No filial altruism
No reciprocal altruism

So I need a sample between individual based evolution and gene base evolution.
For example, many genocide are done against the best and brightest. I wonder what sort of genes encode "kill the best and brightest" and how does that kind of gene survive well in the gene pool?

The prototypical example of this is t, whose existence was predicted by Robert Trivers and featured prominently in the Selfish Gene.  The current dominant point of view in evolutionary biology is that genes act in their own interest and even the 'self' is just a manifestation of the gene's reproductive properties.
t is a locus in some male mice. The t gene characterizes very well the selfishness of a selfish gene.  While most paternal genes are found in 50% of offspring, t is found in over 3/4 of offspring.  Somehow it manages to reproduce at the expense of the other loci in the male mouse.  t itself is not known to convey any advantages to its host organism.  the males tend to be smaller and sometimes even reproductively disadvantaged.
Does that go some way to answering this question?
Animal behavior such as cheating or adopting are often interpretable as reproductive advantages.  Killing the best and brightest definitely are to the advantage of those who are doing the killing.  This SNL Skit has always said it best to me. "... and now I am smart!".  Such kill offs so rarely include killing one's own offspring.  I'm not sure they can be called in conflict with reproductive advantage.  EO Wilson's essay "On Human Nature" does a reasonable case for saying that wars and famines are in fact produced by evolutionary advantage, however cruel and ugly they may be.

And what stops t from dominating and be found in all mice? Oh because it creates weaker male?

you got it.  that's in fact the most famous thing about Selfish Genes - they propagate themselves at the expense of the organism that contains them.  This parasitic case of the gene is not a great strategy but it proves that genes compete directly.  Are there other types of competition?  not everyone agrees :)

Complex behaviours like altruism rarely boil down to a single gene, so it's hard to find a concrete example.
This is one lead:
http://scan.oxfordjournals.org/content/early/2010/10/28/scan.nsq083.full
If you think about it, being altruistic is detrimental to your personal fitness, but inproves the fitness of the population (within certain parameters).
I've only ever heard of clear examples of this in simple game-theoretic simulations.
There may be some good examples here:
http://en.wikipedia.org/wiki/Kin_selection
HTH

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What is an 'ex vivo' experiment?
Simple terminology question:

Is there a hard boundary between in vitro and ex vivo?
Is there a hard boundary between in vivo and ex vivo?

Suppose a sensory neuron is electrically recorded in the following settings:

Surgically accessing through the animal's skin, and patch-clamped,
Tissue surrounding the neuron is almost separated, but still dangling from the larger part of the animal, and patch-clamped,
Same as above, but the slight dangling connection is cut,
The sensory neuron is isolated and put in an artificially oxygenated medium

The sensory neuron is assumed to have purely feedforward signal, and is perfectly healthy in all conditions. Which conditions, if any, are in vivo, in vitro, or ex vivo? (Feel free to propose better hypothetical experiments. I know this example is not the best.)
Am I the only one confused about these terminologies?

It's simpler and broader than that. All depends on the source material for the experiment, if you're talking about experiments: in vivo: animal models
ex vivo: cultured cells
in vitro: purified molecules

The terminologies are there to be helpful shorthands, i.e. they are not mutually exclusive terms and are not strictly defined jargon. Case 1 is in vivo, the rest are not. Concerning Case 2, removing the environment of a neuron cannot guarantee its native state as it exists in the organism, so it is not an in vivo experiment (at least not to most electrophysiologists). In vitro is just a shorthand for where it's taking place. You can simulate an observation on a computer in silico, or make observations on a plate in vitro. If you can't be clear with a shorthand, don't use it!

Ex vivo simply means "outside the normal, living organism", whereas in vitro means "within glass", usually in a cultured system.  They are not exactly same as there is no need for the work to be done in a culture system, although both are not in vivo.  Harvested tissue could be examined ex vivo without being in a test tube, or surgical systems could be tested ex vivo on artificial organ models.  In vivo requires the natural, living organism, although it depends on just how much you are perturbing the natural.
To answer your scenarios,

in vivo
ex vivo, as it is not within the normal organism, although I could understand someone taking issue with that; definitely not in vivo, which is usually for normal conditions only, even including temperature.
ex vivo
If you mean the artificially oxygenated environment of a tissue culture system, then in vitro; if you mean the artificially oxygenated environment of a lab bench with an open $O_2$ valve, then ex vivo would suffice.

Comment to top answer (from Amory): in vivo is not for normal conditions only, half of the times it's not in the "normal" "natural" conditions, that's how you can learn something, you always have to compare a mutated version or diseased version to a healthy control, all is in vivo anyway (when it's in vivo), regardless of the conditions. So in the example 1, are you trying to say that if it is the mouse model for Alzheimer disease, for example, then it's not in vivo anymore?!
Also, "vivo" doesn't mean "living organism", it just means "living".

@kjf That was intended to convey the typical case of a living organism.  Cell culture is living and is an organism, but it is not a "normal organism."  It was intended to be colloquial, as noted where I say "although it depends on just how much you are perturbing the natural."

(Not only) Wikipedia states that in vitro and in vivo have a very hard boundary between them. I something is studied in the context of an organism, without being extracted, it is in vivo, as soon as it is extracted it is in vitro.
Strictly speaking, I would count only the first of your neuron examples as in vivo, because it is still expected to be working like it does inside the organism. As soon as you start having it dangling out, the original "environment" is destroyed.
The relation between ex vivo and in vitro is less defined.
I would count example 2 as ex vivo, but not in vitro, as the Wikipedia article for ex vivo  describes. Same goes for example 3.
Example 4 is of course in vitro. Debatable is, if in vitro excludes ex vivo. It is still taken out of an organism, not grown artificially, so I would still consider it ex vivo.

if you check articles in different fields, chemists and biochemists will usually say cultured cells is in vivo ("the molecules we always study in the tube are now inside a living cell!"), clinical doctors will say it's in vitro ("we take some cells from the patient and put them in a tube"), cell biologists working with clinical samples, animal models, cell cultures, purified molecules, the whole set, will say it's ex vivo ("the molecules that we study are in a living cell, but that living cell that we also study is out of the organism, ex vivo!").

Can you add some reliable references please?

The term in vivo refers to a medical test, experiment or procedure that is done on (or in) a living organism, such as a laboratory animal or human.
The term in vitro, in contrast to in vivo, refers to a medical study or experiment which is done in the laboratory within the confines of a test tube or laboratory dish.
In contrast to in vitro studies, in vivo studies are needed to see how the body as a whole will respond to a particular substance. In some cases in vitro studies of a drug will be promising, but subsequent in vivo studies fail to show any efficacy (or, on the other hand, find a drug to be unsafe) when used within the multiple metabolic processes that are continually taking place in the body.

Please provide a citation or link to supporting evidence. Thanks!


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How stable is in vivo whole cell patch clamping?
For a head fixed animal, how stable is the current state-of-the-art whole cell recording from a cortical neuron? The animal's breathing, and heart beat must move the neuron relative to the recording device quite a bit, and I was wondering how stable the seal would be. How long can one hold a cell, and what is the typical quality of such recording?

After talking to a few electrophysiologists, I found out that they have very diverse opinions.
It seems that a common way of stabilizing the movement of the brain with respect to heart beat is to press the brain with some softish-rigid material like agar. Also, draining the CSF could help but then this cannot be done without sacrificing the animal.
The quality of the patch degrades over time, and it could be held for an hour or more (anecdotal).


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Negative feedback in the fructose metabolism in liver
What happens in liver when large amount of fructose is consumed?
I vaguely remember my biochemistry lecturer telling us that the enzymes that process fructose in the liver does not have negative feedback to control the rate of reaction. But I cannot find a reliable source. How is it regulated if it is?

The main reaction of fructose in liver is phosphorylation, catalyzed by ketohexokinase (UniProt P50053). In one paper with rats it was shown that fructose load can deplete the liver of nucleotide triphosphates (ATP, GTP) although ADP and GDP are inhibitors of the enzyme. This shows there is no effective regulation.
M. I. Phillips, D. R. Davies: The mechanism of guanosine triphosphate depletion in the liver after a fructose load. The role of fructokinase. In: The Biochemical journal. Vol 228, Nr 3, Jun 1985, p. 667–671, {{ISSN|0264-6021}}. PMID 2992452. PMC 1145036.

great answer. thanks. It sounds like an important result, but the paper has only been cited 8 times according to google scholar. I wonder why.

I think it's because most of the work was done with cow's enzyme. Human's enzyme was characterized but no inhibition studies are available for that.


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Does eating antacid (such as Magnesia) before meal have any impact on digestion?
During an anatomy lecture I heard of the importance of the acidity of gastric acid (e.g., killing microorganisms, dissolving food, being a factor for triggering further processes). If a healthy person would eat a tsp. of Magnesia [Mg(OH)2] suspension before a meal (lets say at least 30 min beforehand) and then eat normally, would it have a significant impact on the digestion? Like being unable to extract or produce certain products? Or having a higher risk for a yeast infection?
This question also applies to proton channel inhibitors such as Omeprazole and other drugs like Ranitidine
(Background: The lecture was just a introduction to anatomy for informatics. We never went into greater details.)

A spoonful of NaOH(s) on an empty stomach will have pretty nasty consequences as it will most likely lead to damage to the stomach and internal bleedings. My last concern would be infections or digestive issues.

@ChrisStronks could you explain how it would damage the stomach? I could imagine it would damage the mucus and then the gastric mucosa would be without protection - ready to get a hole, which would be the worst thing I could think of.

NaOH is a strong base and when dissolved is much more harmful to the body than the equivalent of sulphuric acid or HCl and other strong acids. It will dissolve the stomach wall indeed.

If Natron was indeed NaOH (I have forgotten most of my Chemistry knowledge sigh- at least wikipedia seems to say it is NOT- clarifications welcome) then the reaction of high concentration NaOH and HCl is highly exothermic. I can only imagine the disasters it would cause in the stomach.

What language was the lecture in? In German, Natronlauge is a name for sodium lye (NaOH), which will cause extensive chemical burns long before it reaches the stomach. But the word Natron is used for baking soda (sodium bicarbonate), which will not cause immediate wounds. Still, it's not a good idea to eat it, it disrupts the chemical balance of the stomach. I have a relative who, after prolonged use of sodium bicarbonate, got a bad case of GERD, but cannot say if the soda caused it, or if he would have gotten it anyway and the soda failed to prevent it.

@Rumtscho thank you - I am Dutch originally and the Natron is the trivial name for NaOH. You are right. NaOH is a strong base, baking soda is not that bad :)

@Zwie Changed Natron to Magnesia (Milk of Magnesia is used as an antacid). Is it fine?

@WYSIWYG actually I have no idea what Magnesia is. The lecture was in German and I was thinking of Natron as baking soda... kinda assumed it would be the same in English.

@Zwie magnesia is magnesium hydroxide. It is used as an antacid


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Is aquafaba readily digestible?
I have always drained and tossed the water from canned beans. My understanding has been that the water that canned beans are soaked and cooked in contains raffinose, the gas-causing sugars that humans can't digest. This University of Michigan page, referred to in the answer to an earlier question, says:

Rinse beans thoroughly and never cook beans in the water they've
  soaked in. It's loaded with the gas-causing raffinose sugars.

And also advises:

Drain and rinse canned beans. That will get rid of some of the
  gas-causing raffinose sugars (and almost half of the unwanted sodium).

Now that aquafaba is all the rage, though, I'm wondering whether my understanding is wrong. I understand that the bean water would taste mildly sweet and foam up so readily because of the raffinose. But it seems to me that aquafaba would lead to severe bloating and intestinal distress, as it relies for its properties wholly on the gassy and sugary nature of the bean water.
Am I mistaken in my understanding of the need to toss the water that one soaks or cooks beans in? Or is it just that fans consider the bloating and gas a price well paid for the delights of aquafaba?

Hi Verbose, what makes a question "about health" is the knowledge required to answer it. If you ask "is it digestible", then the person answering it would have to know which compounds are digestible, and that would be physiology related knowledge. If you ask "does X contain Y", this is indeed an on-topic question, no matter if you are asking because you think that Y is tasty, or undigestible, or cures acne. The source in your text did list a compound to look for, so I changed the title accordingly.

@rumtscho This question concerned the digestibility of aquafaba. Raffinose is not the only undigestible sugar. The person answering it would have to know what compounds are digestible, but that is not prohibitive. Most questions require an answerer to know the answer. I disagree with the title change and have reverted, I believe it changes the nature of the post too much and was not the author's intent. Note also that this question is specifically not "about health". But if you choose to revert again I will not fight it. Worst case this may be a question more appropriate for the bio site.

(I.e. edit doesn't seem right, migration would seem more appropriate)

@JasonC " The person answering it would have to know what compounds are digestible, but that is not prohibitive" - that is prohibitive, because this is exactly what takes the question into physiology, and makes it out of scope here. I know that the edited question is not equivalent to the original one, but I made the edit because the original one was off-topic. Your suggestion to migrate also seems like a good one, let's try it and see what happens.

I don't see how this is a biology question rather than a culinary question; I'm not interested in the chemical structures of the sugars themselves, nor in the mechanics of digestion. The question seems a natural follow up to the linked question about using the water from canned beans. If that water is inadvisable to use for cooking because of its indigestible and high sodium nature, then that naturally raises a question about its use as aquafaba. And that is a culinary question, not a scientific one. The migration is severely inconvenient

(Whoops, inadvertent send) because it prevents my accepting Jason's answer; even joining Biology doesn't let me do that, oddly enough, and merely clutters up my app feed with bio questions of no interest to me. So yeah, I disagree with the reasoning and the migration.

@rumtscho Well, that could've gone better, lol... Whistles and moseys on home

I think after a while it let me accept the answer, which mitigates some of my sadness at the migration, but I still think biologists would be less interested in this issue than cooks would.

@verbose it doesn't matter if cooks are interested - it is off topic for cooking. We could have closed the question on Cooking without migrating, or answered the partial question of aquafaba containing raffinose sugars, or migrated here. The option "stays on Cooking as it is" was never open, sorry.

@rumtscho you're the boss; it is unfortunate that you would unilaterally migrate the question without seeking off-topic votes from other members of the community, though.

@verbose Fwiw, if it helps: No matter what site it is on, it will still come up the same in web search results. Also Google does place some priority on SE sites, cause ya know, we rock.

Much of it is not readily digestible. According to this chemical analysis:

Finally the results presented here also indicate that the amount of digestible carbohydrates in aquafaba is minimal. For families with GLUT1 deficiency, where a diet with minimal amounts of glucose and carbohydrates digestible to glucose is required, aquafaba can represent an additional foaming or emulsifying ingredient, as an alternative to eggs.

It also contains proteins, presumably digestible. The carb and protein content is significant, according to the study, which implies a fairly decent proportion of undigestable carbs.

@verbose No you are right. I was passing time answering questions during a stressful wait in the animal hospital last night and wasn't all there. I misinterpreted "detectable" as "barely detectable". I can't say for certain just from that article exactly what fraction is undigestable carbs but it suggests a significant portion. Also cursory research does suggest public opinion that blaming farts on the family dog is not a big price to pay for the many wondrous and magnificent delectations of aquafaba, or at least, nobody seems to complain about it or contest it.

I mean, you could just drink it for a few days and see what comes out. For science.

Yes, for science and keep a laptop by your bedside, and write copiously as quantities so of undegestable carbohydrates entering your large intestines and bowls are fermented by bacteria. We wish you luck and god speed.  And remember, you do this so that others will never need to experience the same tragic horror. For Science.

@JasonC I'm sorry to report that the "Honey, we need to get a family dog so I can try out aquafaba. For science" ploy didn't work. I'm still pet-less.


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What prevent us from synthesizing a cell from "scratch"?
In this recent article "Design and synthesis of a minimal bacterial genome", they created a minimal cell with only 473 genes. However, they didn't synthesize all of the necessary components of the cell to get going. How much of those extra stuff can we currently synthesize, and what are the ones that we can't synthesize in the lab to create a working cell from scratch?

You mean ribosomes and other cellular organs by "necessary components"?

It is nearly impossible for even nature to create a cell from scratch (this might be debatable). Note that new cells in organisms are made from pre-existing cells via cellular division, and almost all necessary substances are provided to new cell from parent cell only.

Designing/engineering functional proteins from scratch is feasible but would take a ridiculous amount of work. Michael Hecht is one person I know of who has done this (example: http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0015364). J Craig Venter wants to determine the minimal set of molecular machines required for life, regardless of their origin. This would begin to illuminate which protein functions are absolutely required for life. I believe we could design and engineer proteins de novo to perform all of those functions in theory, but in practice it would be unrealistic. I think the key point to glean from their synthetic cell is the set of functions, not specific genes, required for life. Those functions could be performed by evolved or synthetic molecules.
As a side note, while I believe that to be the overarching goal, they do note the surprising lack of functional characterization in the syn3.0 genome.


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Is there any biological reason for humans to rest after x days of work?
A lot of people live in a 7 day cycle, where 5 days of work are followed by 2 days of "rest". Vacations and holidays can increase the time available for resting.
Is there any biological reason for humans to rest after x days of work? Can this be explained by the buildup of by-products of metabolism, like Homocysteine?

No, it's explained by Family, a very complex stuff :)

Don't forget, humans rest every day when sleeping. Whether you are at work or not, your body is still active, still "working".

Sleep is circadian in nature, with this question I'm trying to understand if there are other, longer term cycles that I don't know of yet. The wikipedia article on circa-septan cycles is a stub: http://en.wikipedia.org/wiki/Circaseptan

The 7-day week is a recent innovation. The Romans used an eight day week. The Incas used a ten-day week. I don't know of any evidence that prior to the last ten-thousand years people followed anything other than solar (daily and annual) and lunar (monthly) cycles.

7-days weeks is specific to the cultures tracing back to traditions in the Mediterranean region (Judean, Roman, Egyptian, etc.) and is mainly preserved in Chistian and Arab cultures. Other cultures had weeks of different length or in some cases no days of rest at all (Japan?). Note also that there may be more psychological rather than physiological reasons for taking a day of rest - certainly in the Jewish tradition.

I think your main assumption is not true -- we do not really rest on weekends.
I do not know many people who only rest on weekends. Usually this is the time for socializing, doing home-related work, physical activity (sports, hiking), spending time with your kids, all of which is often almost the opposite of a rest.

This is what I'm trying to find out - if there's a reason to take it slow once in a while and relax,  purposefully not working. I'm sure with the pace of modern life people cram their weekend to the brim and then wish they did not have to sleep. But this does not appear to me to be the nature's way. I can't think of an animal that is as active at all hours of a day as humans are

My point is that with few exceptions, weekend is not the time to "take it slow and relax".

@Alex, Sheep spend 15% of their time sleeping, the rest is either active foraging or chewing cud.

Our bodies work in cycles.. Circadian cycles that refer to our daily routines. We need sleep every night, eating at night affects us differently than if eating in the morning, etc... all because our bodies functions work in these 24 hour cycles...
Now, beside the circadian cycles, our bodies also have circaceptan (or 7 day) rythms. For example, our immune system picks every 7 days (reason why the 7th day after a transplant is the most likely one may reject the organ). This 7-day cycle is also observed in other life forms...
But, read for yourself:
 a seven-day cycle has been found in fluctuations of blood pressure, acid content in blood, red blood cells, heartbeat, oral temperature, female breast temperature, urine chemistry and volume, the ratio between two important neurotransmitters, norepinephrine and epinephrine, and the rise and fall of several body chemicals such as the stress coping hormone, cortisol. "In fact," Perry and Dawson note, "weekly rhythms appear easiest to detect when the body is under stress, such as when it is defending itself against a virus, bacterium, or other harmful intruder. For example, cold symptoms (which are really signs of the body defending itself against the cold virus) last about a week. Chickenpox symptoms (a high fever and small red spots) usually appear almost exactly two weeks after exposure to the illness.:" [Susan Perry and Jim Davson, The Secrets Our Body Clocks Reveal, (New York: Rawson Associates, 1988) p. 22.]
Doctors have long observed that response to malaria infection and pneumonia crisis peaked at seven days. Organ transplants face similar crises as the body's immune system attack the foreign organ. Campbell explains: "When a human patient receives a kidney transplant, there is a rhythm of about seven days, a predictable rise and fall in the probability that the body's immune system will reject the new kidney. A major peak of rejection occurs seven days after the operation, and when a serum is given to suppress the immune reaction, a series of peaks occurs, with increasing risk of rejection, at one week, two weeks, three weeks and at four weeks, the time of the highest of all." [Jeremy Campbell, Winston Churchill's Afternoon Nap, (New York: Simon and Schuster, 1986). p. 76]
So, in answer to your question... it makes a lot of sense that if so many of our body functions have 7-day rythms, we would feel a biological need to slow down and allow our bodies to rest, rejuvenate....

Searching for "circaceptan rhythms" didn't yield so many results. Do you have any additional references?

Did the doctors discover the circaceptan (7 day) cycle on people who live in the modern system of a 7 day working week? Because it would be funny that they found a 7 day circadian rhythm in people who have a 7 day week. And the animals in that study, did they live near to cities that operate and pollute in 7 day variables, or were these animals out in the proper wildernesses where humans don’t venture too much and so there is a chance the 7 day human week isn’t smelting buried into their bones by us?


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How do neurons form new connections in brain plasticity?
I've been reading about brain plasticity and how the brain can "rewire" itself.
One of the things that is not clear to me - how neurons can establish new connections. Does this rewiring mean that neurons can "disconnect" from other neurons to reuse existing dendrites?  Or do neurons grow new dendrites to make these new connections, adding to existing ones?
Thank you for your input!

Related (almost a duplicate): http://biology.stackexchange.com/questions/8/under-what-conditions-do-dendritic-spines-form

I haven't read anything particularly about dendrites being reshaped, though I would expect them to be as flexible as other parts of the cells.
The more commonly discussed topic (in my literary experience) is reshaping of the axon's branches before forming synaptic terminals. These branches are not fixed even in adults - neurons can grow new and retract old branches, attaching (synapsing) to other cells in new places and removing old connections (see Wikipedia: Synaptogenesis).
Additionally to this actual change in the number of synapses, individual synapses can be regulated in their signal strength by adjusting the number of neurotransmitter receptors in the postsynaptic membrane (Gerrow&Triller, 2010, also see Wikipedia: Synaptic plasticity)

There is actually quite a lot of literature on changes dendritic branching.

Must be because my "reading" is mostly that suggested by lecturers and syntactic elasticity was a topic in lessons but dendritic wasn't :)

Take for instance this (but there are many other examples) Chronic Psychosocial Stress Causes Apical Dendritic Atrophy of Hippocampal CA3 Pyramidal Neurons in Subordinate Tree Shrews

Like @nico says, there is a lot of literature on dendritic "reshaping" (see, for instance, http://biology.stackexchange.com/q/8/72). I'd say that dendritic reshaping is discussed with about as much frequency as axonal reshaping.

Under conditions where the sensory input to the cortex has been altered, large-scale changes in dendritic branching have been observed after enriched environment experience (e.g. Greenough and Volkmar, 1973) and sensory deprivation (e.g. Tailby et al. 2005). However there is a discrepancy between those post-mortem studies and more modern in vivo studies, where only small-scale changes of the dendritic tips have been observed (Schubert et al. 2013). Large-scale axonal re-arrangements have been also implicated in post-mortem studies after sensory deprivation (e.g. Darian-Smith and Gilbert, 1994) but no such effects have been convincingly described in living animals.
These somewhat different results may be due to several technical limitations: 1) post-mortem studies have utilized injectable tracers and their uptake and/or expression levels may contribute in part to the observed results, 2) in vivo studies have been confined to superficial dendrites due to optical access limitations. Furthermore, these manipulations of the animal's experience are relatively crude ways of probing cortical circuits and do not necessarily reflect the processes that the brain may use under more physiological conditions.
By far the most well described processes under enriched environment experience, sensory deprivation and sensory stimulation, and learning paradigms are microscopic changes at the level of individual synapses. These include increased synaptic turnover (Trachtenberg et al. 2002, Xu et al. 2009), synaptic stabilization (Holtmaat et al. 2006), and synaptic strength changes (Hofer et al. 2009). These microscopic changes provide a very economical way, in comparison to large-scale dendritic or axonal re-arrangements, in which neuronal circuits can be extensively rewired given the enormous potential of dendritic spines and boutons to sample different synaptic partners (Stepanyants et al. 2002).

References
Darian-Smith, C., & Gilbert, C. D. (1994). Axonal sprouting accompanies functional reorganization in adult cat striate cortex. Nature, 368(6473), 737–740. https://doi.org/10.1038/368737a0
Greenough, W. T., & Volkmar, F. R. (1973). Pattern of dendritic branching in occipital cortex of rats reared in complex environments. Experimental Neurology, 40(2), 491–504. https://doi.org/10.1016/0014-4886(73)90090-3
Hofer, S. B., Mrsic-Flogel, T. D., Bonhoeffer, T., & Hübener, M. (2009). Experience leaves a lasting structural trace in cortical circuits. Nature, 457(7227), 313–317. https://doi.org/10.1038/nature07487
Holtmaat, A., Wilbrecht, L., Knott, G. W., Welker, E., & Svoboda, K. (2006). Experience-dependent and cell-type-specific spine growth in the neocortex. Nature, 441(7096), 979–983. https://doi.org/10.1038/nature04783
Schubert, V., Lebrecht, D., & Holtmaat,  a. (2013). Peripheral Deafferentation-Driven Functional Somatosensory Map Shifts Are Associated with Local, Not Large-Scale Dendritic Structural Plasticity. Journal of Neuroscience, 33(22), 9474–9487. https://doi.org/10.1523/JNEUROSCI.1032-13.2013
Stepanyants, A., Hof, P. R., & Chklovskii, D. B. (2002). Geometry and structural plasticity of synaptic connectivity. Neuron, 34(2), 275–88. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/11970869
Tailby, C., Wright, L. L., Metha, A. B., & Calford, M. B. (2005). Activity-dependent maintenance and growth of dendrites in adult cortex. Proceedings of the National Academy of Sciences, 102(12), 4631–4636. https://doi.org/10.1073/pnas.0402747102
Trachtenberg, J. T., Chen, B. E., Knott, G. W., Feng, G., Sanes, J. R., Welker, E., & Svoboda, K. (2002). Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex. Nature, 420(6917), 788–94. https://doi.org/10.1038/nature01273
Xu, T., Yu, X., Perlik, A. J., Tobin, W. F., Zweig, J. a, Tennant, K., … Zuo, Y. (2009). Rapid formation and selective stabilization of synapses for enduring motor memories. Nature, 462(7275), 915–9. https://doi.org/10.1038/nature08389


text:
In C. elegans, why does knock-down of cco-1 in some tissues increase lifespan, and knock-down of cco-1 in other tissues decrease lifespan?
Full question: In C. elegans, why does knock-down of cco-1 via RNA interference in specific tissues like body wall muscle decreases life span, whereas knock-down in the nervous system and intestine increases life-span?
http://www.wormbase.org/db/gene/gene?name=WBGene00000371;class=Gene
Also from paper 'The Cell-Non-Autonomous Nature of Electron Transport Chain-Mediated Longevity'
Supposedly, it's through hormesis. But why is there a differential effect from tissue to tissue?

Let us first answer the question: what is the effect of cco-1 knock-down in C. elegans. Durieux J., Wolff S. and Dillin A. in their paper "The cell-non-autonomous nature of electron transport chain-mediated longevity". Cell 144:79-91 (2011) provide the following explanation:

The life span of C. elegans can be increased via reduced function of
  the mitochondria; however, the extent to which mitochondrial
  alteration in a single, distinct tissue may influence aging in the
  whole organism remains unknown.

So, by slowing-down the mitochondria function you manage to slow-down the normal wear-and-tear of the mitochondrial enzymes, which are normally responsible for the so-called respiratory chain -- one of the fundamental biological process for storing energy in cells (despite the common belief, ATP is rarely to store energy, but mostly to transfer it to the target!).
If we group the tissues where the knock-down leads to similar effects we will get muscle cells opposed by neurons and intenstine cells.
This is just my suggestion, but if we approach those groups with the idea of energy storage and re-use in mind, we'll see that muscle cells have much higher turnover of energy (for contraction and even more for dilation), whereas nerve and intestine cells are not that dependent upon energy and have much lower baseline of energy consumption and turnover.
Now we can suggest that cco-1 knock-down decreases the functions of mitochondria in muscle cells below the acceptable level and the decreased life span is due to depletion of mitochondrial energy storage under light stress conditions which would have been easily coped with under normal conditions. And vice versa, nervous/intenstine cells requiring a constant energy source at a much lower level just help the animal to cope with stress more effectively even during old age, leading to increased life span.


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Does muscle growth trigger angiogenesis?
So heavier people generally have more blood than lighter people (this is why heavier people generally need to take higher doses of medication for the same effect of medication). They also have more blood to draw from. But this fails to differentiate between muscle mass and fat mass.
So here's my question: Is the net angiogenesis per gram of muscle more or less than the net angiogenesis per gram of fat?

Without having an exact proof, I'd definitely say yes. Muscles need more nutriments to function than fat, and if you look at the body builders you'll see that some those guys have thicker brachial veins than an average aorta.

See this article as to why your underlying assumption is incorrect.  Higher doses must be given due to the high fat solubility of some drugs as well as differences in liver and kidney size.

Oh wow - thanks for that information. Doesn't that mean that for those with high muscle mass (relative to fat mass), that the guidelines for how much drug they take might be inaccurate?

Specific doses are usually not calculated as several factors may dictate upgrading the dose, not only weight or fat content. Drug sensitivity can be caused by the speed at which the liver removes the drug from the blood or genetic factors that make the drug more effective or the severity of the condition for instance.  Weight is just one factor that will increase the dose, all other factors being equal.

Muscle is heavier than fat so actually it does. Muscle growth directly increases angiogenesis. Particularly the mechanism relies on hypoxia, any tissue with less than adequate blood supply secretes factors which result in angiogenesis. This is seen frequently when muscle is bulking as this increases the oxygen requirement but is also seen in heart muscle where subsequent to a heart attack tissue which isn't dead but mildly suffering caused new blood vessels to grow to supply this tissue (as the other ones are blocked which was the cause of the heart attack in most cases).


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Is there any ethnicity difference in average pupil size?
I have seen some articles claim African Americans have smaller average pupils than whites while Asians have larger pupils. Is it true?
link:
Y. Li, D. Huang; Pupil Size and Iris Thickness Difference Between Asians and Caucasians Measured by Optical Coherence Tomography. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5785.

if you've "seen some articles", it would be good to cite them/give links in your question ...

Welcome to Biology.SE! Please take the [tour] and then go through the help pages starting with [Ask] questions effectively on this site and [edit] your question accordingly. In particular, we expect you to demonstrate that you have done research on your own and then, informed by what you have learned, ask any questions you still have (ideally with references to reliable sources). ——— In this case, I suggest finding and providing references to any primary research that supports these claims. Thanks!

Please also include the complete reference information since links can (and do) break. One easy way to get that information is to search for the paper on Google Scholar and click on the ‟ symbol to get reference information. This is a good example of how to format references.

Note nowhere does it say they controlled for height, which is not equally distributed. A control for different heights would be important. A comparison based on latitude for a developmental effect would also be useful.


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Evidence against aging caused by loss of information in genome and epigenome in humans?
After reading Lifespan by David Sinclair I'm curious if there is any evidence against aging being caused by loss of information in genome and epigenome of 3.7*1013 cells in human body.
Specifically has anyone compared the genome and epigenome of the same humans at different ages?

It's important to mention the alternate hypothesis of not loss of information but different information. Taking the telomere shortening example, that is straightforward loss, but that DNA isn't informational really; rather it plays a structural role in the cell such that when it's lost it gets hard to segregate chromosomes. The other mutations and epimutations aren't (necessarily) loss of information, but on average the substitution of different information and rearrangement of the existing information.

By loss of genome, I think he is referring to the shortening of telomeres. This
study compares the length of telomeres of human epidermal cells. The authors report that over time, the telomeres do shorten. They also report on the rate of loss of around 32 base pairs per year. Thus there is loss of some genomic information over the age of an individual.
However, the loss of epigenomic information is trickier. The primary reason for it being some regions of the genome have been shown to lose DNA methylation marks whereas others gain it. However, leaving that aside, there is comprehensive proof that DNA methylation changes with age. This study on female peripheral leukocytes shows that approximately 350 regions (known as CpG islands) which are differentially methylated with age. Of these 350, 250 loci become hypermethylated i.e. methylation increases with age while the opposite happens for the remaining 150 loci.

Thanks for the explanations. Very helpful! In the 1st paper when the scientists use 13.8±1.0 kb, does kb mean kilo bytes of base pair ATCGs so 1 kb = 1000 bytes = 8000 bits 0s && 1s representing ATCG... where A&&T = 00, T&&A = 01, C&&G = 10, && G&&C = 11 ?

@Wang-Zhao-LiuQ The kb in biology means kilo base pairs. So 13.8 kb is 13800 base pairs. In some publications the unit kbp or bp is used to avoid confusion.

Thanks! I think I should rephrase the question is aging caused by the change of genetic information? Losing 32 base pairs or gaining 32 base pairs could both contribute to expressions of genes that cause signs of aging.

Your second sentence is correct. However, keep in mind that it is not entirely clear whether loss of telomeres causes aging or is a result of aging. For example, mice have short lifespans but less degradation of telomeres compared to humans. You may want to explore some literature on how metabolism affects aging process as well.

From what I know, I would expect that no one has performed a similar experiment to what you propose. Sequencing a genome is still rather expensive and inaccurate. Some parts of the DNA can’t be sequenced at all and most methods still rely on comparing the sequencing reads to a reference genome, making these kinds of predictions harder.
Getting snapshots of an epigenome (chromatine modifications, transcription factors) to the accuracy that you can detect a notable difference after a few years is even harder.
Also, by analyzing the genome of a given somatic cell, the cell is being destroyed and not available to test it again after a couple of years to see if relevant mutations occurred.
In short: Sinclair‘s predictions are probably hard to verify at this point.

Have any ideas on how to analyze the genome of a somatic cell without destroying it? Can it be cloned perfectly && then we destroy the cloned cell for analyzing?

This runs into a similar problem: cloning a cell involves removing it from the tissue. I’m not an experimentalist though. Maybe it’s achievable with a cell culture. Using a living organism you would have to come up with something novel, probably.


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Nucleotide Sequence
So i am from an IT and my sis who is doing medicine asked for my help on this question i have no clue where to start actually. Please Help. So the question is:

What is the nucleotide sequence for vibrio cholerae bacteria or
treponema pallidum bacteria? or any other related bacteria?

After the nucleotide sequence is got, is possible to use BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to align and compare a query DNA sequence with a database of sequences?
Please correct me wherever i'm wrong and thanks for helping

I think this is more suited to SE.bioinformatics.

Yes it is possible. That's what BLAST is for. The nucleotide sequences can easily be found by searching the NCBI Nucleotide database.

There are at least 102 genomes of the bacterium Vibrio cholerae that have been sequenced. For example, Vibrio cholerae 01 biovar El Tor strain N16961 has two chromosomes spanning 4,033,464 bp in which its ORFs code for 3,827 proteins and 122 RNAs. Check here The DNA is available at NCBI as entries AE003852 and AE003853.
There are at least 11 sequenced genomes of Treponema pallidum. For example, Strain Fribourg-Blanc has 1,004 protein genes and 54 RNA genes distributed over 1,140,481 bp genome. NCBI DNA sequence entry NC_021179.1
I suppose one may align multiple sequences of whole genomes if that's what intended, but I do not advise it unless one is ready to curate the results. Instead, I recommend studying a given open reading frame using its DNA sequence, or better yet its corresponding protein sequence. BLAST should be quite helpful in finding similar sequences to compare to.


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How do short polypeptide chains serve as identifiable antigens?
My understanding is that MHC class I and II bind to relatively short polypeptide chains (~ 20ish amino acids). I'm surprised that a non-self protein fragment that short would be distinctive enough to target for an immune response. Also, how does expressing one polypeptide chain help the immune system identify the source? Isn't it quite likely that the presented polypeptide is in the interior of a protein and would never actually be exposed to the environment until after it's been lysed? Or is it hydrophilic peptides that bind?


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Role of Bone marrow in T- lymphocyte development?
After I have read this statement in my textbook :
"Both bone-marrow and thymus provide micro-environments for the development and maturation of T-lymphocytes."
I have been searching for some detail on internet but I had found that only thymus is involved in maturation of T-cells while bone marrow in involved in maturation of B-lymphocyte.
So I'm bit of confused what exactly is the case .

T cells arise in the bone marrow, as that's where the common lymphoid progenitor cells are (they also give rise to B cells). However, their development and maturation - TCR gene rearrangement, positive selection, and negative selection - occurs in the thymus1.
I suppose that one could say there is a micro-environment for developing T cells in the bone marrow, as that's where they originate, but pretty much all development takes place in the thymus. Your textbook's statement is misleading at best, although I would say it's just wrong.
It's worthwhile to note that some mature T cells do take up residence in the bone marrow as part of immune surveillance, and micro-environments do form with other cells as part of that, so perhaps that may be what your book is referring to.
Reference

Kumar BV, Connors TJ, Farber DL. Human T Cell Development, Localization, and Function throughout Life. Immunity. 2018;48(2):202-213. doi:10.1016/j.immuni.2018.01.007


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What are all the possible developmental stages an insect can be in?
I'm making an app which allows the user to report insect observations. Many bugs may exist in different forms throughout their life time. For example, a butterfly will exist as an egg, a catterpillar, a pupa and lastly as a butterfly. A greenshield bug can live as a nymph and then as an adult.
I want to make one list that contains all morphological stages that insects can be in. Then the user can select the appropriate one for the species he or she observed. My question is: What are the names of these stages? Are there any other stages next to the egg stage, caterpillar/larval stage etc?
Thank you

The stages are called instars:
https://en.wikipedia.org/wiki/Instar
It changes depending on the insect: it can be caterpillar, grub, maggot, 2nd instar, 3rd instar, larval stage, larva, eruciform, scarabaeiform, campodeiform. Here are some pages:
https://entomology.ca.uky.edu/ef017

This process is refereed to as Metamorphosis, is a biological process by which an animal physically develops. Furthermore, Animals can be divided into species that undergo complete metamorphosis ("holometaboly"), incomplete metamorphosis ("hemimetaboly"), or no metamorphosis ("ametaboly").
Otherwise there is the process known as Holometabolism, which more specifically pertains to insect development. This process defines almost all the types of insect development.
Others include: Ametabolism and Hemimetabolism.

can a nymph also be considered a holomatebolic stage?

A nymph is the immature form of some invertebrates, particularly insects, which undergoes gradual metamorphosis (hemimetabolism). So unfortunately no.


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Why genes are assumed to follow multivariate normal?
I wonder why gene expression data are very frequently modeled by multivariate normal distributions. What is the reason for those strong assumptions that the genes follow multivariate gaussian? Are there any reasons specific for genetics other than the reasons for general gaussian assumptions (the ease of calculation, etc.)?

Usually if something is not expected to behave according to some scheme then the measured values for such a parameter are assumed to be normal. It is not just with gene expression but with all types of measurements like dimensions of an object, luminosity of an electric bulb, range of a bullet etc.
In any measurement, the random error is modeled using normal distribution. I don't have a very intuitive explanation for why random errors follow normal distribution but mathematically it comes from the central limit theorem.
Now each gene is a variable and measurement of each gene suffers some random error; so a multivariate normal distribution is used.
When we dismiss a null hypothesis in a t-test or z-test what we are actually doing is dismissing our parsimonious notion that a sample is drawn from a given normal distribution. This means two things:

The sample belongs to some other normal distribution (different $\mu$ and $\sigma$)
The sample follows some other distribution

But a t-test will never be able to point out the exact reason. All it tells you is that the sample is not from some given normal distribution.


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Is post-transcriptional regulation of gene expression an epigenetic process?
Is post-transcriptional regulation of gene expression (for example regulation by microRNAs) a type of epigenetic gene expression regulation?
I think we can categorize it as epigenetic since the DNA sequence is not changed, but I have never come across that terming in any papers. Does someone have any idea, or know of any papers that categorize post-transcriptional regulation as epigenetic?

miRNAs and other post-transcriptional regulators are very well "genetic". They are encoded by genetic elements, are expressed and are affected by mutations. Just because this mode of regulation was not well known previously, it should not be classified as an epigenetic mechanism while the traditional protein based transcription factors (TF) are not.
Epigenetics, as it is originally defined (the "formal definition") is about mechanisms that can perpetuate the state of a cell to its next generation. Inheritance of gene expression programmes is therefore epigenetic. Although the gene expression programmes themselves can be implemented via different factors including protein and RNA based regulators, they would not necessarily constitute the epigenetic mechanisms that lead to inheritance of this state.
rg255's point of view is that any mechanism that causes a variation in the functional aspects of the genome without altering the genome sequence itself, would be epigenetic. This is technically correct but in that case all gene expression regulators including TFs should constitute epigenetic mechanisms.

Now, the main issue is where to draw the line between gene regulation and epigenetics?
In my opinion, the epigenetic mechanisms are one of the ways to regulate the gene expression. Although histone modifications and DNA methylation regulate gene expression and also confer heritability to the gene expression programme, the heritability can be implemented without them as well.
You can imagine a cell as a vessel which runs a system of biochemical reactions. This system can have multiple steady states (for e.g. multiple fates of a stem cell). To perpetuate a state, the new cell just needs to have the right initial conditions. This can be proved mathematically too. Such a system can be implemented via the traditional transcription factors as well. So what is epigenetic?
IMHO epigenetic was a loose term to denote something that people were not fully aware of, at that time. Anything that was not directly mediated by transcription factors was termed as epigenetic, including long distance regulators, non-coding RNA etc.

BOTTOMLINE
I would not classify non-coding RNAs as "epigenetic" for the very reason that they are encoded by genes and have more or less a direct effect on the target genes, just like TFs (which are apparently not epigenetic). As for the papers, there were many papers that used to assign these under epigenetic mechanisms, but that is IMO just too vaguely arbitrary. (Ironically, I happened to come across miRNAs and lncRNAs while I was doing a summer project on epigenetics and was reading relevant papers.)
What should be considered epigenetic would be a subject of another debate.

How can you implement functionally non-fatal heritable mechanisms without the presence of histone modifications and methylation? The conservative epigenetics definition revolves around heritable mechanisms (not loose), the contemporary epigenetics definition is now loose encompassing fields which we understand better than we did in previous years (noncoding RNAs, chromatin architecture included).

How can you implement functionally non-fatal heritable mechanisms without the presence of histone modifications and methylation?... It is theoretically possible and there are examples too but I cannot remember them at this moment. The point is that you are thinking from a top-down point of view (looking at the world from a human/animal/eukaryotic point of view). A self sustaining system does not need histone modifications. Look at the cell cycle, for example.

I meant an experimental system. I would be very interested in seeing such an implementation in a eukaryotic system where the mechanisms have been completely removed (not cases such as ipsc where the marks are procedurally removed and re-established by the system) and the system is able to propagate without these mechanisms.

@KoustavPal That would be very difficult to implement because the cells have evolved to be dependent on that complexity and it would not be possible for the cell to survive if you strip that off from it. However, a self sustaining system can exist without these mechanisms (exist just because of the GRN). It is likely that histone modifications etc came later in the course of evolution. Prokaryotic organisms don't even have typical histones!

GRN?. Ofcourse they had to come later, you might find this on prokaryotic memory interesting, I read something similar or this one a long time ago (http://www.nature.com/nature/journal/v460/n7252/abs/nature08112.html), my point with the original question was a system which has evolved a regulome around these features would find it very hard to survive without them, although a system without them wouldn't need them at all. So that sort of makes the statement moot. We are talking about very different systems altogether, with entirely different contexts, the statement to me implies

that a system which has these mechanisms may still have them taken out and yet survive and lead to fully functional progenies

@KoustavPal GRN = gene regulatory network. We can talk in chat. Getting too long for comments.

I think WYSIWYG's explanation and mine are more in tune with the consensus than that of rg255. I highly doubt you will find many molecular biologists who think of miRNA's as epigenetic

@WYSIWYG Thanks for the reply. Could you please explain a little more why it is apparent that TFs are not epigenetic?

@user5054 People somehow don't consider them epigenetic. It is just arbitrary. There is no logic.

On epigenetic and genetic effects:
Changes to the genome can be of two key types: genetic and epigenetic. Genetic changes are those which cause changes in the nucleotide sequence. Epigenetic are changes to the genome that do not involve making changes to the nucleotide sequence, e.g. post-transcriptional processing.

"Functionally relevant changes to the genome that do not involve a
change in the nucleotide sequence. Examples of mechanisms that produce
such changes are DNA methylation and histone modification, each of
which alters how genes are expressed without altering the underlying
DNA sequence".

Epigenetics is also generally used to refer to the study of variation induced by heritable non-genetic factors that affect the genome, such as maternal and paternal effects. The two subtly different definitions are responsible for some of the common confusion.

"Today, epigenetics refers to the study of heritable changes in gene
expression without the change in gene sequence. ".

On microRNA
There is some contention around whether miRNA is specifically an epigenetic mechanism - you've used it as an example - post transcriptional modifications would generally be considered epigenetic effects. See the paper from which the following extract comes which covers "classical" mechanisms too:

"Whether miRNA regulation is an epigenetic mechanism in its own right is unclear"

Also see whatisepigenetics.com:

"At least three systems including DNA methylation, histone modification and non-coding RNA (ncRNA)-associated gene silencing are currently considered to initiate and sustain epigenetic change."

And part of the conflict is perhaps because miRNA's are seemingly involved in the control of epigenetic processes:

"Epigenetics is defined as mitotically and meiotically heritable changes in gene expression that do not involve a change in the DNA sequence. Two major areas of epigenetics—DNA methylation and histone modifications—are known to have profound effects on controlling gene expression. DNA methylation is involved in normal cellular control of expression, and aberrant hypermethylation can lead to silencing of tumor-suppressor genes in carcinogenesis. Histone modifications control the accessibility of the chromatin and transcriptional activities inside a cell. MicroRNAs (miRNAs) are small RNA molecules, ~22 nucleotides long that can negatively control their target gene expression posttranscriptionally. ....
Taken together, miRNAs can be considered important players in the epigenetic control of gene expression."

From a quantitative geneticist standpoint, if it affects phenotypic variation by altering genomic properties but there is no variation in the DNA sequence, then miRNA based post-transcription modification is a source of epigenetic variance. It seems that, for molecular biologists, post-transcription modification by miRNA falls outside of the classical definition of epigenetic effects, but I've not seen any literature explaining why nor classifying it as a genetic effect.

The point is not that the field of molecular biology has a different definition. What I want to point is that if miRNAs are considered epigenetic then why not TFs? As per your definition TFs/spilicing regulators/RNA binding proteins/riboswitch/protein kinases etc should also be considered epigenetic.

I disagree with rg255 on this. Most if not all of posttranscriptional modification is encoded in the actual DNA sequence. Those microRNAs for example can be determined by reading the DNA bases or finding the encoded enzymes that do RNA editing (like C to U by TPR enzymes). The DNA sequence already encodes all the information that will determine if it will get modified or not.
Epigenetic regulation on the other hand are encoded on the histones and other proteins associated with DNA and can by definition not be understood by reading the sequence (e.g. acetylation and methylation). This information does not code for proteins or transcripts.
EDIT:
A quick Pubmed search underlines what most scientists think is epigenetic Pubmed search. You see loads of methylation/histone research but miRNA or transcription factors are nowhere to be seen.

the regulation of a gene's expression is an epigenetic process, regardless of whether the ability to do such regulation comes from genetic information

The expression of a gene is modified post transcription, not because a nucleotide was changed elsewhere in the genome, but because that nucleotide exists

e.g. expression of gene X may be regulated to be differently expressed in two cell types, each cell has the same sequence of DNA, but one part of that DNA gives instructions about regulation of gene X in specific cells - there are no differences in sequence between the cells, but there is a difference in expression, therefore it is epigenetic

@rg255 then the TFs are also epigenetic. Every gene expression regulator is epigenetic that way.

if it creates variance in the genome, but does not come from variance in the sequence, it's epigenetic

the genomic variation is not generated by sequence variation - the machinery to create that variation can be coded within the sequence, but the genomic variation is not created by the existence of that segment of sequence

@rg255 Your point of view is correct but then that would mean that any mechanism of gene expression regulation arising because of the network dynamics should be considered epigenetic. I would agree with you on this definition but the very scientific community that works on these aspects considers only a few cases.

Just to note on your pubmed search, methylation research has a considerable headstart on miRNA regulation research having been discovered decades earlier.

Let us continue this discussion in chat.

You should check out this article by Adrian Bird, titled Perceptions on Epigenetics
Excerpts from the article:

Should heritability be mandatory in a contemporary view of epigenetics?

...

To explain why, it is necessary to introduce a third, somewhat informal, ‘definition’ of epigenetics that has crept into widespread use. This incarnation of epigenetics encompasses the biology of chromatin, including the complex language of chromatin marks (see page 407), the transcriptional effects of RNA interference (see page 399) and, for good measure, the effects of the higher-order structure of chromosomes and the nucleus (see page 413)

Finally he goes on to propose a new definition:

The following could be a unifying definition of epigenetic events: the structural adaptation of chromosomal regions so as to register, signal or perpetuate altered activity states

If we consider that this definition might also be a possible definition for what epigenetics is, then yes RNAi would fall under the broader bounds of epigenetics


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Does vitamin D form crystals in the blood with calcium and magnesium?
I read on a vitamin-D product (2000NE) that I should not use calcium and magnesium with it, because it can result crystal formation in the blood. I googled, but find anything about this. Is it true?
(Somehow I doubt it, but I don't know the exact mechanism how vitamin-D works by building bones. Afaik. there are osteoblasts and osteoclasts which constantly build and degrade the Ca-Mg-PO4 depos, and I assume vitamin-D is just a cofactor of an enzyme or it is required to produce a hormone. I never heard of Ca/Mg - vitamin-D cross-reactions...)

Since vitamin D leads to the absorption of calcium, it can lead to hypercalcemia and calcium precipitation! This is true specially in patients with hyperphosphatemia (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2486454/)

Vitamin D does not bind to Calcium. It is a pre-hormone which is converted to calcitriol. Calcitriol has an intracellular receptor (VDR) that, as a heterodimer with Retinoid-X receptor, regulates gene expression.
I guess the "should not be had together" is because of possible over-absorption of Calcium. There are commercially available supplements (e.g. 1, 2) that have Vit-D + Ca + Mg.

Additionally both are present in milk, if I remember correctly.

The exact words were "it forms crystals in the blood with calcium or magnesium" (in Hungarian ofc.). I guess it was just a bad joke of a frustrated worker, I don't know...


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Can a stereo microscope be used for mycology?
I have a need for a stereo microscope to do electronics work. I also have a strong desire for a stereo microscope to do mycology. I would like to be able to,

Clearly identify mycelium, from bacterial contamination (no interest in bacteria).
Be able to isolate spores of mycelium.
Identify dikaryon, from monokaryon
Ideally, but not required be able to identify different types of mycelium that are undesirable (mold).

Could I take a trinocular stereo microscope and add a barrow lens that increases the zoom and decreases the focal length, and use it for biology as desired above? Some stereomicroscopes that have 2x Barlow advertise 225x, like the AmScope ZM2225NT.
What would the downsides of this approach be?

I'm not sure, but I suspect the answer here is a general "no".
The reason I say this is that stereo microscopes typically do not have the magnifications needed to see the mycelium and other structures needed for ID of fungi clearly. For this you need a minimum of 100x (total magnification) and ideally 400x - 1000x magnification (that's 10x, 40x and 100x objective lenses on a compound microscope respectively). The one you mentioned from AmScope should just be capable of this range with a maximum magnification of 225x. I don't know if that magnification is with a Barlow fitted or not.
Fungal ID is typically done by staining the hyphae and fruiting structures, mounting on a slide, and then observing these via transmitted light - brightfield illumination, with the light shining from the bottom, directly through the object. Some stereomicroscopes will have this capability, but definitely not all. Those that do won't have a condenser, which converts the point illumination of the bulb into a parallel or converging beam, which is needed to provide even illumination of the right angle for the objective lens being used so that you can see the object clearly.
Stereomicroscopes would be useful for observing a fungal colony from above, but this isn't often all that useful for identification purposes.
Barlow lenses will help provide more magnification, I think at the expense of depth of field, though this shouldn't be much of a consideration for mounted specimens. Nikon's Microscopy U has lots of really good information on stereomicroscopes, including Barlow lenses (they call them "attachment" lenses) that you might find useful in determining for yourself.

Some stereoscopes do have condensers, for example, the Nikon Diascopic Stand that I have for my SMZ-U, has a condenser. It is a rarer feature for sure, but even some of amscopes offerings have it

for some things sure, like looking at surface structures in more detail, and with 200x power you might see micro structures including perhaps cells. but most fungal microscopy is done with a compound microscope of 400-1000x power, which allows you to clearly see cells and cell types, hyphae, and spores


text:
How can you explain the origin of life out of nonliving matter (abiogenesis)?
According to biochemist Robert Shapiro, the "primordial soup" theory is as follows:

Early Earth had a chemically reducing atmosphere.
This atmosphere, exposed to energy in various forms, produced simple organic compounds ("monomers").
These compounds accumulated in a "soup", which may have been concentrated at various locations (shorelines, oceanic vents etc.).
By further transformation, more complex organic polymers – and ultimately life – developed in the soup.

From: en.wikipedia.org/wiki/Primordial_soup
I don't understand how in step four life forms out of nonliving organic polymers. Could you explain this process? Can it be reproduced in the lab?
Louis Pasteur already disproved Spontaneous Generation: The law of biogenesis

I think my 'answer' to this question may give you something to think about: http://biology.stackexchange.com/questions/19720/how-is-the-origin-of-life-out-of-non-living-matter-explained-by-evolutionary-the Also, I'm sure this is a duplicate of many questions that have been asked here before. The simple answer is: we don't know.

Btw, the life-history tag indicates questions related to life-history traits and not with the "history of life".

Welcome to Biology.SE!
Your question has nothing to do with evolutionary biology
Evolutionary Theory does not explain the origin of life just like the Theory of Gravity does not explain the diffraction of light! In other words, explaining the origin of life is not within the scope of evolutionary biology. You should edit your title. But still, your question is within the scope of Biology so that your question is totally on-topic. This question would probably also be on topic on Chemistry.SE.
Some other posts may interest you
You may want to look at other posts on this site. This post for example explains what are our issues in order to select which of our hypotheses really explain the origin of life. This post, this one may eventually interest you as well.
Your question might be too broad. You may want to start with some introductory readings
Abiogenesis is not my field and I know very little about the origin of life. But at first sight, I am a bit afraid that your question is too broad and inaccurate and a good answer would ask for writing a lot. This post will give some good source of reading to further your knowledge. It may be good to start there and come back to this site with a more accurate question.
Hope I could help you a bit.

Some might think that this is a chemistry question, and indeed most of the work is done in chemistry departments, but that isn't because its not biology.
As far as evolutionary theory, there is a well-established definition of life as self-replicating systems and very little else.  Richard Dawkins' theory of the Replicator  is not always completely accepted (the link is to Stephen Gould's essay, who was usually in the opposition to Dawkins until he passed away).  Nonetheless, its logic is hard to completely dismiss: Once and only once chemical systems can self replicate then evolution happens - selection, variation and replication.  Dawkins thought springs from the pretty justifiable position that DNA is the center of all inheritance and variation in living things.  I'm not going to get into the arguments for and against but suffice to say that the replicator model might not be entirely true, but its not entirely wrong.
As far as the fourth step you mention, there are theories which are very credible but its reasonable to say that we are short on details... they may not satisfy and they certainly have not duplicated step 4...  The most detailed and credible theory is the RNA World Hypothesis.  At some stage, RNA polymers were able to self-replicate, pretty much by themselves it seems.
The evidence for RNA world is compelling but its more molecular archaeology than a series of coordinate events as we see it.  The ribosome and other RNA genes show that RNA could be fundamental genes.  In particular the ribosome consists of RNA whose structure are supported by protein, but RNA does all the chemical work.
Consider the odds.  The prebiotic soup may have existed for the better part of a billion years and covered all or a good part of Earth during much of that time.  As such, its exceedingly unlikely that we will be able to reproduce self replication of RNA in the lab.  Lucky and intelligent guesses may produce an environment that is just barely pre-biotic and produce a replicator, but even that seems quite unlikely.  Almost all this work tries to show that compounds and processes that come close to life are possible in pre-biotic Earth.


text:
What is the typical amount of full length clones in a cDNA library?
For a Yeast Two Hybrid project we need to make a cDNA library from a single celled organism. We used the Cloneminer II kit from Thermo. We followed the manual and performed all the quality control steps and everything checked out. However out of curiosity we sent 10 individual colonies for sequencing and while all of those give hits in our organism only 40% seem to be even approaching full length in the coding sequence (80-90% coverage).
Is this normal or did we have poor input material/made a mistake? What is the average number of full length clones for a typical cDNA library?


text:
Is there a place or tool from which I can find or generate genome data for epistasis detection?
I am busy researching a school project for my honors in Electrical Engineering. The project is to create a deep neural network to detect epistasis. I have very little knowledge in the bioinformatics field and while I have managed to pick up most of the theory on epistasis and all of the new terminology, I still lack knowledge of the resources that are available to someone in my position.
I will be needing a large amount of data to train my neural network. I do not mind what form the data is in I just need many examples of epistasis. Where can I find high quality, easily accessible data for this kind? Or alternatively, what tools can I use to generate this kind of data?

What specific kind of data are you looking for? What research have you done so far?

@MattDMo What I'd like is data that takes the form of a vectors of As, Ts, Cs and Gs, one for each chromosome perhaps. I'm not 100% sure though. I have looked at a tool called GAMETES which generates SNP based models for simulation studies and can also be used to generate data, however the data isn't quite in the form I just described. I have also had a look at PLINK, however, the data it generates is in linkage equilibrium which as I understand it means that it won't be of use for epistasis detection because epistasis occurs when there is linkage disequilibrium.


text:
Alleles always occur on the same locus
My book says "alleles of the same gene always occur at the same locus, but on a pair of homologous chromosome"
Please how is it possible since genes of particular character always occur on the same chromosome? When we know that alleles combine to form a gene, how comes then do they occur on homologous chromosome?

An allele is a "version" of a gene - you don't combine alleles to make a gene. I strongly suggest you do some more research on your own, perhaps starting here.

Homologous chromosomes refers to the "twin" chromosomes in your cells (one from your mother and one from your father). Each somatic cell has at least one nucleus containing all your chromosomes*2. Having two sets of all chromosomes is referred to as diploid. On a chromosome, genes are encoded through different nucleotide sequences in DNA, and thus have a specific spot on the chromosome. The spot where a gene is located is called the locus, and because you have two chromosomes in your cells that contain the same genes (though maybe different alleles!), the genes will be in the same locus (same spot) on the both of the chromosomes.
The alleles do not actually combine to form a gene. An allele is simply one variant of a gene, and there can be many alleles for one gene. If a person has the same allele on both chromosomes they are considered homozygous for that allele and if they have different alleles they are considered heterozygous for it. Genes can also be considered either dominant or recessive. If they are dominant, they can be expressed with just one copy in the cell, but if they are recessive the same allele must be present on each chromosome. The alleles do not "combine to form a gene", they are simply separate copies of the same gene with possible variations on them. Homologous chromosome simply means the other copy (e.g. a gene on the 3rd chromosome of your mother will be present on the 3rd chromosome from your father although it could be a different allele due to a variation in nucleotide sequence). I hope this helps, although I would suggest doing some more research on your own, perhaps through https://www.khanacademy.org/science/biology. Their videos tend to be very descriptive.

@Shirouh If this answer addressed your problem, please consider accepting it by clicking on the check mark/tick to the left of the answer, turning it green. This marks the question as resolved to your satisfaction, and awards reputation both to you and the person who answered.


text:
How can an enzyme favor even chained fatty acids?
If we look at text books for the human fatty acid metabolism (FA synthesis or β oxidation), they typically shows even length fatty acids (C8, C10, etc). Now, from a chemical perspective, I am wondering how the involved enzymes recognize that the fatty acid is an even number of carbons.
Can someone point me to (historic) experiments that have been done that show that these human pathways indeed only apply to even chain length fatty acids? Or do they also act on odd length fatty acids?

The first experiment which showed that fatty acids are oxidized in C2-units has been done by Georg Franz Knoop and been published 1904 as "Der Abbau aromatischer Fettsäuren im Tierkörper.". The paper in reference 1 states:

Georg Franz Knoop discovered fatty acid β-oxidation. In 1904, he
  published his classical experiments using odd and even chain ω-phenyl
  fatty acids such as ω-phenylvaleric acid and ω-phenylbutyric acid
  (Knoop 1904). Knoop fed these compounds to dogs and analysed their
  urine. In dogs that had been fed the odd chain fatty acids, he found
  hippuric acid (conjugate of benzoic acid and glycine), whereas, the
  dogs that had been fed even chain fatty acids excreted phenaceturic
  acid (conjugate of phenylacetic acid and glycine). From this he
  concluded that the metabolism of fatty acids proceeds by the
  successive removal of two carbon fragments. The remaining fatty chain
  had to contain a carboxylic acid. He postulated that oxidation took
  place on the β carbon atom, an oxidation unknown to organic chemistry.

If the fatty acid chain has an uneven number of carbon atoms, the resulting propionyl-CoA is modified into succinyl-CoA which is then used by different processes.
During the breakdown of the chain, Acetyl-CoA groups are released (at the end of each step), which can then be further metabolized by the body. Acetyl-CoA is fed into the Krebs cycle and then oxidized.
References:

A general introduction to the biochemistry of mitochondrial fatty
acid β-oxidation
Fatty Acid Oxidation

Good answer, which can be summarized as "they don't". I would add that for synthesis, there is also no specific odd/even check, they are just synthesized two atoms at a time. Though there are fatty acid modifying enzymes that have a particular length preference (that's why we tend to see particular chain lengths)

Victor, indeed, it shows they do not. OK, that's half of the answer. What about the FA synthesis pathway? Does that also not have a preference?

The basic answer is that the synthesis enzymes start with a 2-carbon molecule and then build the chain 2 carbons at a time. If you introduce unusual enzymes that can start with a 3-carbon molecule, you can get odd-chain fatty acids. However, the terminating enzymes do have some specificity in terms of chain length preference. https://en.wikipedia.org/wiki/Fatty_acid_synthesis


text:
Can hepatitis B also infect vaccinated persons?
If a person is vaccinated against hepatitis B: Is there any chance that he also gets the infection by physical contact? Is it a congenital disease or not (if the parent is hepatitis positive is the offspring is also affected or not)?

I have edited your question to make it better understandable, feel free to roll back the edit if it not appropriate.

It is possible but the chance is somewhat low - 10%.
You can check to be sure that the level of surface antibody in your blood is efficient by a simple blood test.
The Hepatitis B vaccine is very effective at preventing Hepatitis B virus infection. After receiving all three doses, Hepatitis B vaccine provides greater than 90% protection to infants, children, and adults immunized before being exposed to the virus.
In highly endemic areas, Hepatitis B is most commonly spread from mother to child at birth (perinatal transmission), or through horizontal transmission (exposure to infected blood), especially from an infected child to an uninfected child during the first 5 years of life.
Find more about this here:
http://www.who.int/mediacentre/factsheets/fs204/en/
You can find all about Hepatitis B vaccine on the CDC's website:
http://www.cdc.gov/vaccines/vpd-vac/hepb/public/index.html


text:
Choosing the best assay for my experiment?
Which of the following assays can be used to determine whether cell death specifically due to apoptosis has occurred in a given cell sample? Choose all that apply.

WST-1 Assay
Caspase Colorimetric Assay
Trypan Blue Staining

Which of the following assays can provide direct information about the plasma membrane integrity and/or morphological features of apoptotic cells? Choose all that apply

Annexin V-FITC / Propidium Iodide staining.
WST-1 Assay
Caspase Colorimetric Assay
Trypan Blue Staining
Wright Staining

My knowledge is very limited with these assays. I know Trypan Blue is used to measure a cell's viability, with dead cells stained blue. Wright Staining is used to study the cell's morphology and makes fragmentation visible. Caspase color and the WST assay seem very similar to me. Both measure cell viability with spectrophotometry.

Welcome to Biology. Homework-type questions are welcomed, provided OP shows prior efforts to answer the question in the post. Please edit.

I'm really not provided much information on the assays and the internet is not being very consistent. As far as I know for the first one I believe Trypan Blue Staining is one but im not sure which of the other two could be considered... possibly Caspase colormetric assay? As for the second question i think it is wst and wright staining. Again my knowledge is very limited with these assays. But I would really appreciate clarification or guidance.

I know trypan is used to measure a cells viability, with dead cells stained blue. wright is used to study the cells morphology and fragmentation is visible with it. caspase color and WST seem very similar to me. Both measure cell viability with spectrophotometry. otherwise i cant differentiate.

Great additions, please edit this info in the question, as folks may not read the comments. After edits I'm happy to retract my close vote and give you an upvote too :)

I'm voting to close as homework, despite the editing it still makes very little effort to self help.

I agree with @rg255. You should at least make an attempt to read what these assays are. "Caspase color and the WST assay seem very similar to me": this is not true. Read about them. As it stand it looks exactly like a homework with a very little effort.

Necrotic cells could be trypan blue positive. WST-1 Assay could reflect cell number by measuring mitochondrial dehydrogenase, therefore, you could not know how many cells die in dishes. When you define apoptosis in your experiment as caspase dependent cell death, measuring caspase activity is the best.
PS
Trypan Blue Staining actually is a method to see membrane integrity.


text:
How to search for organisms by criteria such as growth substrate and overflow metabolites?
I'm looking for a database or a systematic way to search for organisms which meet certain criteria. My criteria descending importance are:

growth substrate
chemolithoautotrophy (on which substrate)
overflow metabolite
methanogen
Nitrogen-fixation

Is there a database / some other means to find this information other than manual digging?
I tried NCBI taxonomy, LINNAEUS, uBio, KEGG, organism-tagger and others, but these don't appear to be capable of this kind of query. They all seem to require the organism name to begin with. But maybe I'm missing something?
thanks for any hints.
Ingvar

Some of the criteria, I guess are difficult to find (Also for my lack of understanding of those terms). For the rest you can search these databases:-

Growth substrate: I don't really know what you are asking here — growth rate in minimal media/optimal growth medium ? Nevertheless, see the Media Database
Methanogenesis: Ortholog Table - KEGG MODULE M00567
Nitrogen fixation: Ortholog Table - KEGG MODULE M00175


text:
Why re-label the strand ends in 3' DNA labelling?
I have a problem with a molecular biology question; I don’t understand how DNA 3’ labelling works.
I took a diagram from my lesson and tried to understand with it; this is what I understood.
If I’m wrong, please tell me and correct me.

We take a restriction enzyme which will cut both strands. The bottom strand will be a 5’ end. So, the top strand is shorter than the bottom strand.
We will add a Klenow fragment which, with its polymerase activity, polymerizes in the 5’->3’ direction the missing part of the top strand, with dNTP α 32P (labelled by a radioactive phosphate in the alpha position).
We get two strands which have the same size, of which one will be strongly labelled.
We finish with a terminal transferase which will catalyse the addition of dNTP α 32P on the 3’OH ends of both DNA strands (in order to label these ends).

But, if I’m right, could you explain the necessity of the last step; why label both ends since we have already labelled the top strand?

Welcome! It will help if you explain the purpose of labeling the DNA according to the lesson: Are you creating a radioactive probe for in situ hybridization? For Northern blotting? Is this a RACE assay?

The goal of this label is not defined ; it is just in the part "how to label DNA " ?   I see that "in situ hybridization", "Northem blotting" are quoted after this part.

Are you asking why it is necessary to label both strands in the final step versus just labeling the bottom strand, or are you asking why it is necessary to label both strands versus labeling only the top strand?

There are many ways to label nucleic acids for probes (Summary of Methods).
While there are cases where Klenow fragment-mediated fill-in should sufficiently label both strands of your probe, there are many others where this would not be enough.  Here are two illustrative, although somewhat contrived, examples:

Example 1: Double digest with 5' and 3' overhangs
  Imagine you did a double digest with BamHI and KpnI to release your probe sequence from the plasmid in which you propagate the sequence.  BamHI will give you a 5' overhang, but KpnI gives a 3' overhang.
Resulting BamHI & KpnI double digest fragment:
  5'-    GATCCNNNNNNNG      - 3'
             |||||||||
  3'-        GNNNNNNNCCATG  - 5'

Subsequent treatment with the Klenow fragment + $\alpha$P32-dATP + cold dNTPs will both fill-in the 5'-overhang (Klenow's 5'->3' polymerase activity) and remove the 3' overhang (Klenow's 3'->5' exonuclease activity).
Post-Klenow reaction (bold = new bases, * = radio-labeled nucleotide):
  5'-    GATCCNNNNNNNG      - 3'
         |||||||||||||
  3'-    CTAGGNNNNNNNC      - 5'
           *

At this point only one strand is radio-labeled so the terminal transferase step is needed to add a label to the top strand.

When using $\alpha$P32-dNTPs, you often pick only one base to be radioactive (eg dATP as above).  Sometimes you also just spike the reaction with radioactive dNTP leaving a population of cold dNTPs to be used most of the time.  In either case you now have the chance of not incorporating only non-radioactive bases.

Example 2: Fill-in reaction that fails to incorporate $\alpha$P32-dATP:
Imagine a digest with PspI and want to fill-in the 5' overhangs using $\alpha$P32-dATP + cold dNTPs.  PspI recognizes CCWGG sequences where W is either A or T so that some fill-ins will incorporate the $\alpha$P32-dATP, but others will not.
Possible digests and fill-in reactions:
   5'-  CCTGGNNNNNNNNNNCCTGG - 3'
        ||||||||||||||||||||
   3'-  GGACCNNNNNNNNNNGGACC - 5'
                 |
         Digest with PspI
                 |
                 V
   5'-  CCTGGNNNNNNNNNN      - 3'
             ||||||||||
   3'-       NNNNNNNNNNGGACC - 5'
                 |
        Fill-in with Klenow + hot dATP + cold dNTPs
                 |
                 V
   5'-  CCTGGNNNNNNNNNNCCTGG - 3'
        ||||||||||||||||||||
   3'-  GGACCNNNNNNNNNNGGACC - 5'
          *

Again you are left with only one strand being labeled (or no strand labeling if the sequence was 5'- CCAGGNNNNNCCTGG - 3') and a need for the terminal transferase step.