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Why does autosomal trisomy have more adverse effects than sex choromosome monosomy or trisomy?
Autosomal trisomy syndromes are usually lethal but sex chromosome number related abnormalities do not lead to that much fatality. why?

To support mdperry's answer: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2429870/ Figure 2 in particular should draw your attention as it shows the previously mentioned correlation between chromosome size, transcripts and embryonic viability. Apologies, I would have added this in comment form but I do not have enough reputation.

It has to do with dosage compensation:

The Y chromosome contains relatively few genes. Interestingly, chromosome 21 and 18, which are over represented in the most common viable trisomies, are also relatively small.
Humans already possess a mechanism to compensate for additional X chromosomes: X-inactivation. Normally this inactivates one of the two X chromosomes in females but can also inactivate two in cases of X trisomy.

Humans are much more able to tolerate extra sex chromosomes than extra autosomes. … Compared to autosomal trisomies, … sex chromosome trisomies are fairly benign. Affected individuals generally show reduced sexual development and fertility, but they often have normal life spans, and many of their symptoms can be treated by hormone supplementation. The ability of humans to tolerate supernumerary sex chromosomes is quite remarkable, as individuals can survive with as many as four sex chromosomes. This tolerance most likely relates to both X inactivation and to the small number of genes on the Y chromosome. In fact, when cells from individuals with more than one copy of the X chromosome are analyzed under a microscope, all but one of the X chromosomes appear as condensed Barr bodies, the cytological manifestations of X-chromosome inactivation. Supernumerary copies of the Y chromosome may be tolerated because the few gene products of the Y chromosome are not required for survival.

From: Chromosomal Abnormalities: Aneuploidies by Clare O'Connor (emphasis added)

An added bonus (and I feel as though I should actually write something myself), I recently read a paper studying trisomy 21:
Jiang J, Jing Y, Cost GJ, Chiang JC, Kolpa HJ, Cotton AM, Carone DM, Carone BR, Shivak DA, Guschin DY, Pearl JR, Rebar EJ, Byron M, Gregory PD, Brown CJ, Urnov FD, Hall LL, Lawrence JB. 2013. Translating dosage compensation to trisomy 21. Nature 500(7462):296-300.
Using zinc finger nucleases, they inserted a XIST transgene into chromosome 21 of cells derived from Down syndrome patients. XIST is a gene centrally involved in X-inactivation. They found that, by inserting it into chromosome 21, they were able to silence one and revert the trisomic cells to a more normal phenotype.

Somewhat related: what-effect-does-the-barr-body-have-in-relation-to-female-turner-syndrom

First it depends on the species in question. I assume that you are referring to humans and/or mice. In diploid organisms the toxicity of losing or gaining a chromosome is related in part to the size of the chromosome, but also determined by which genes are on the chromosome in question. Some genes could be lethal if their copy number is increased or decreased (dose sensitive). The longer the chromosome the more genes it is likely to have and therefore the greater the chance of a problem. In humans viable trisomics usually involve the shortest chromosomes. Human X is very large but it is dosage compensated.

This is an interesting answer. A reference or two would cheer things up.

@AliceD: Thank you for the feedback. I am just learning to use markdown, and so thus far my answers and comments lack formatting. I am also limited to using mobile devices when composing, and have not mastered the skills required to cut and and paste images and URLs between Apps. My working hypothesis is that individuals posting questions here are familiar with search engines (and so any references I insert would be largely redundant). Therefore I limit my posts to material that might not be easily found on the Internet. I have also updated my profile to explain my knowledge base.

Yes folks will probably be aware of how to use google, but without references answers are generally scrutinized and not upvoted, or even downvoted. Everyone can write up unfounded fancy stuff. The art of answering is to give folks the opportunity to do further reading and confirm the answer. That's how this site works. The fact that half of your answers remains not voted upon supports this notion. Don't generate your own working hypotheses. The help center is a good source of info.

The morbidity is high because autosomal trisomies affect somatic / autosomal chromosomes, which are essential to the process of life. In fact, the majority of fetuses affected by autosomal trysomy are miscarried, with a few exceptions (Down syndrome is one). The sex cells have only half the number of chromosomes,23, of which the affected ones are usually the XX or XY. Their functions are different and although tremendously important, they are not vital, people can survive and live normal lives with these disorders: X, XXX, XXY, XYY. Mader, chapter 24.

X chromosome contains many vital genes.

Assuming you are discussing our species, sex chromossomes abnormalities do not lead to such fatality because of it's purpose, i.e a gene in these chromossomes mainly affects sexual related hormone producing which do not play any role (there are exceptions like steroid hormones) in general metabolism or imune responses so a mutation in those genes or a duplication of chromossomes does not cause the death of the individual and it survives during the embryonic and fetal stages becoming later a healthy individual but with serious sexual problems like infertility and sterility . In the other hand, a mutation or duplication in autosomal chromossomes is likely dangerous to survival of the organism and many times it cannot survive in the early stages. Those who survive many times face serious health problems in their life because unlike sexual activity, autosomal gene functions are necessary for cells to survive. Sexual activity is just a way to pass genes between generations not to survive in one generation !

the fact that a trisomy 21 results in serious mental and physical disabilities proves that "i.e a gene in these chromossomes mainly affects sexual related hormone producing...] is not true.


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Why does gaining a hydrogen in biology considered reducing a molecule?
I've learnt in chemistry that gaining electrons means reduction, while losing electrons means oxidation. But why is it in Biology textbooks I sometimes come across the term gaining hydrogen??

An example would be nice. It's all about context. And they are confusing terms. An oxidizing agent gets reduced when oxidizing something else etc.

Additionally I think this topic is better suited at chemistry.stackexchange.

No only biology, this notion is there in organic chemistry as well. Remember reduction of alkenes? One of the most common 'reducing agents' used in organic chemistry is LiAlH₄

So in a system of two compounds participating in a redox reaction: an oxidizing agent accepts electrons, and thus the oxidizing agent becomes reduced, and the other compound which loses electrons becomes oxidized. A reducing agent donates electrons, itself becomes oxidized, and reduces the other compound. In lots of biological reactions, you can stylize the gain or loss of hydrogen as the gain or loss of an electron, because hydrogen generally donates its sole electron to any given bond.

The definition of electron acceptance and donation came later. Previously oxidation was just addition of oxygen and reduction meant removal of oxygen/addition of hydrogen.

Hydrogen can accept electrons or donate them, so it can get a bit confusing. For instance, hydrogen donates electrons from a reducing agent. For example, in biology, consider cellular respiration. A co-factor will carry electrons often by reducing an atom with a nitrogen.
Consider NADP+ to NADPH.

The nitrogen atom on the Pyridine group has an oxidation state of +1. When it picks up a hydrogen, to become NADPH, the oxidation state will go to 0 by gaining electrons brought by the hydrogen. Although the hydrogen doesn't bond to the nitrogen directly, it breaks the double bond on the para carbon (The carbon on which the new hydrogen is attached) at the top of the ring, and thus distributes the electrons to the nitrogen.
EDIT -
Hydrogen comes from some reducing agent, not a free hydride I'm sure.

Are you able to annotate the drawing of NADP so that I can follow your explanation better? Thanks :)

I agree with @Teige - a reaction instead of a single molecule would help.

There is no Hydride-Anion present in any living cell.

Oxidation state and charge are not the same thing. The pyridine in NAD+ is positively charged, but that does not mean that it is +1 oxidation state.

source: Stack Exchange

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

sort: votes


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What's the difference between male and female?
As long as we only look at humans the differences are clear: males have chromosomes XY, produce sperm and don't get pregnant. Females have chromosomes XX, produce egg cells and bear babies. But when you consider other species, things are more complicated: in birds, it's females who carry the Y chromosomes. In pipefish and sea horses, it's the males who get pregnant. So maybe the only reliable criterion to tell if an animal is a male or a female is looking at its reproductive cells, and decide if they look like spermatozoa or egg cells. But then there are male and female plants, where none of the methods above applies.
So how do you tell, in general, who's male and who's female in a species? Or is the distinction arbitrary?

Sexes (male and female) are generally defined in terms of Anisogamy, which means that there are size differences between the gametes (i.e. the reproductive cells that fuse at fertilization). The sex with smaller gametes is defined as male and the sex with larger gametes is defined as female, and individuals that can produce both types of gametes are called hermaphrodites. This is the case in both animals and plants (plants have pollen vs. ovules and microsporangia vs. megasporangia), and the definition of sexes is therefore not dependent on chromosomal makeup as such or which sex that carries the young. Actual sex determiniation in a particular species is often based on chromosomal inheritance though, but other systems also exists (e.g. environmental cues or sequential hermaphroditism). Anisogamy also comes in several different types, where animals generally have Oogamy, where males have small mobile spermatozoa and females large stationary egg cells.
More generally, Anisogamy is a special case of mating types in sexually reproducing organisms, where fertilization (on syngamy) generally can only occur between gametes of different mating types. In for instance fungi, gametes of different mating types are of the same size (isogamous) and they are therefore only labelled as mating types (e.g +/-) and not different sexes. Functionally, if we are simplifying the issue, male gametes can be viewed only as carriers of genetic information, while female gametes also contain the nutrients necessary for the early development of a newly formed embryo.

In general, It's pretty much the reproductive cells with plants and animals.  The male's unique cells (sperm, pollen) fertilizes the female's unique cells (egg, ovule).  The resulting offspring develops and hatches from within the female's egg/ovule.  Now whether that egg/ovule stays put, gets ejected, hides in a flower, or transferred to the male (like your seahorses)... that's just rearranging the furniture after it's been designed.

You may think of the penis and vagina as well. In animals, males certainly have penises and females have vaginas, huh? As it turned out, this is not always the case. In This Insect, Females Have Penises And Males Have Vaginas. You can read more about it here.

So as fileunderwater answered, the criteria for differentiating between male and female is the size differences between the gametes. The sex with smaller gametes is defined as male and the sex with larger gametes is defined as female.

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Obvious phenotypic inheritance markers in humans
In humans, are there phenotypic markers that prove parenthood? For example, if mother and father have <trait characteristic> (let's say, specific ear lobe form), then any child will have <trait characteristic>. If the child does not exhibit that characteristic, than either father or mother or both are only alleged, not actual.

Interesting question. Eye and hair colour (two "obvious" ones), to my knowledge, can be different from either parent due to the dominant/recessive nature of alleles. I would be interested to know of any "true" , as you put it

I've previously heard claims that a cleft chin is such a trait, but this seems to be a myth: udel.edu/~mcdonald/mythcleftchin.html. The trait supposedly has a strong heritable component though. Blood type could sometimes be a strong indication of parenthood (e.g. an O father should normally not produce AB offspring), but not always.

Not that I know of.

No. To determine the genetic relationship (if any) between two individuals requires molecular evidence based on genetic markers in their somatic DNA. Using phenotypes could be misleading because a genetic marker (an allele) can have variable penetrance (the percentage of carriers who actually have the phenotype) and variable expressivity (the severity or degree of the phenotype). Some of this variability can come from gene-environment interactions, and some could come from epigenetic effects (ie non-heritable).


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Edman method to identify peptides with Phenylisothiocyanate (PTH)
We all know that in this method the PTH reacts with the first amino acid (aa) from the N-terminal to the peptide and separates from it giving PTH-aa so that we can know the amino acids sequence in the peptide.
The question is what prevents the PTH from reacting with the second amino acid after it separates with the first amino acid in the same time and phase? Because if it does we can't distinguish between the first and second amino acid residue in the sequence.

Welcome to Biology SE! Just to improve your wording here - if we all knew what Edman did you wouldn't need to explain. Don't expect everyone to be a Biochemist working on proteins. also - prevent acronyms like aa. Don't apologize. Just ask the question.

And isn't the answer linked to the chain termination methods in DNA sequencing like the Sanger method? http://en.wikipedia.org/wiki/Sanger_sequencing

The short answer is that the Edman degradation is a multi-step process. The Wikipedia page has a decent picture of the mechanism. In practice, the peptide is reacted with phenylisothiocyanate (PTH) under mildly basic conditions to give a thiourea, which is stable. The excess PTH is separated from the thiourea intermediate. The thiourea is then treated with acid to cleave the N-terminal residue from the peptide. The PTH is no longer present, so there's no concern about reacting with the second residue.


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Are unilaterally deaf people able to determine where sound comes from?
My question is on people deafened in one ear, but normal hearing in the other.
Time and level differences between the two ears are only part of how the human body can localize the source of the sound. So what other factors are there and are they enough to help accurately determine the location of the sound for unilaterally deaf people ?

The ear (including the external flap) can do quite a lot of directional determination via phase-angle measurements. It's not like the  eyeball, which measures only intensity.

A good friend of mine is deaf in one ear. He can usually tell where sound comes from based on volume and how muffled it is. But also, if the sound is on his bad side, he often doesn't hear it at all. So I think he may just be assuming that whatever he hears is on the good side. He swears that he can tell, though. He does get it wrong from time to time too

From what I could gather, there is a decent amount of variability in the ability to detect which direction sound is coming from in unilateral hearing impaired people. It would appear that some people have severely impaired localization skills, akin to someone with an earplug in one ear, but others can use their monaural hearing cues to better locate sound.
Monaural cues seem to be based on the structure of the outer ear as well as how the sound bounces off of the head and shoulders.
http://www.sciencedirect.com/science/article/pii/0378595594900531
http://en.wikipedia.org/wiki/Sound_localization#Monaural_cues

Short answer
Sound localization in the vertical plane should not be affected too much, while localization in the horizontal plane is impaired in unilateral deafness.
Background
The two most important mechanisms used to localize sounds can be grossly divided into localizing sounds in the horizontal (azimuth) and vertical plane (elevation).
In the horizontal plane it is the exquisite capability of the auditory system to distinguish between slight intensity, latency and phase differences between the two ears that allow the brain to deduce where a sound is coming from. For low frequencies it is mainly the inter-aural time and phase differences that play a role. For high frequencies, the difference in sound level between the ears becomes more important (head-shadow effect). For example, if a sound is coming from the left, the left ear will receive the highest sound level. The sound stimulus will also have a shorter latency because of the intervening head tissue, and a phase difference due to the time lag (Van Wanrooij & Opstal, 2007).

Head shadow effect at high, but not at low frequencies. Source: University of Edinborough
In the vertical plane and front-back discrimination it is the analysis of spectral shape cues that lie at the basis of directional hearing. The spectral cues arise from direction-dependent reflections within the pinna (described by so-called head-related transfer functions, see below figure graphs). The pinna provide a monaural localization cue for sound frequencies exceeding about 3–4 kHz (Van Wanrooij & Opstal, 2007).

Head-related transfer function due to the pinna. Source: University of California
However, this hard distinction between elevation and azimuth directional analysis is somewhat simplified, as it appears that elevation is also analyzed in terms of binaural cues, and azimuth is also identified using monaural cues. Hence, all available cues seem to be integrated for both vertical and horizontal localization. Nonetheless, the two distinct pathways are the primary and preferred ways of the auditory system to determine elevation and azimuth. However, when one ear is plugged, or in unilateral deaf people, the textbook binaural hearing in the horizontal plane may be substituted by monaural cues, and I quote from (Van Wanrooij & Opstal, 2007):

[L]isteners rely on monaural spectral cues for sound-source azimuth localization as soon as the binaural difference cues break down.

Therefore, to wrap up, unilateral deafness degrades directional hearing and in particular in the horizontal plane. However, the use of monaural cues may still allow localization of sounds in the horizontal plane in unilaterally deaf subjects, albeit less effectively than normal-hearing subjects using binaural hearing (Van Wanrooij & Opstal, 2007).
Reference
- Van Wanrooij & Opstal, J Neurophysiol 2007;97:715-26

While this is a very good answer, it ignores our ability to move our heads. While for very short sounds we cannot move our heads fast enough, for longer sounds, head movements should introduce a host of monaural cues.

@StrongBad - yes certainly, this answer pertains only to the situation where the head is fixed. Of course, when folks are allowed to move their heads or move toward a  source they will definitely find it through intensity cues based on trial and error. This answer deals with short tone bursts and fixed head position.


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Explaining natural selection in moth
Industrial melanism is an example of natural selection. Can it be considered as a evolution due to anthropogenic action?

Evolution is a change in allele frequency through time. Alleles underlying moth melanism are changing through time in response to environment change, so indeed moths are evolving.
The environmental change is induced by human activities indeed. But the question of what is the cause of the environmental change causing the selection pressure is totally irrelevant to the question of whether there is such selection pressure.
Artificial selection is in essence the exact same thing than natural selection except that there is a goal (increasing milk production or making smaller dogs for example) that is decided by humans. In the moth example, the environmental changed is caused by human activities, but it has never been a goal for human to put selection pressure on moths to force them to change color. As a consequence, change in color melanism is an evolutionary process that is induced by natural selection, selection toward a new environment.
Evolution is not just the same thing as natural selection. The reason is that other mechanism than natural selection yield to change in allele frequency. One of them is genetic drift. You can have a look to this post to have a better understanding of what genetic drift is.
Hope that helps.

Thanks. I got it. Your post on why genetic drift occurs in small isolated population made it better for me to understand it's occurrence in small population

You're welcome. If I answered your question, you can check the answer. Otherwise, you can point to the elements that are still unclear to you.


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How do birds deal with their eyes drying out at high speeds?
My eyes tear up when cycling at 15 mph, which is nothing compared to bird airspeeds.
Do birds continuously produce lots of tears and blink a lot, or do the eyes self-moisturize from the inside without need of blinking, or are their eye surfaces just dry most of the time and they don't mind?

I realise that this isn't the point of your question but wearing glasses may solve your cycling problem.

I do wear glasses. I imagine the tearing would be much worse without them.

Birds have a body part known as the nicitating membrane otherwise known as the "third eyelid". This part has become vestigial in humans, where it remains as the plica semilunaris.

This image of a masked lapwing clearly shows its nicitating membrane in action, where it covers the eye in a horizontal motion. This is analogous to blinking in humans, and the membrane moisturises the eye and removes debris while the bird is in flight.

This paper explains in detail how the peregrine falcon (the world's fastest bird) clears and moisturises its eyes when diving at speeds of over 300km/h.

The paper you cited also mentions that most birds' eyes have, in addition to the lacrimal gland, an extra secretory gland at the base of the nictiating membrane. At least in falcons, this secretes a fluid that is more viscous and resistant to evaporation than thinner tears. The nictiating membrane then spreads this over the eye on each blink. The membrane also has microvilli lining the inside of the leading edge that scrubs away the old layer of this viscous fluid, and any tiny debris that got stuck in it since the last blink.

Amphibians also have this membrane to help them visualize underwater @SuperElectric

Birds, like amphibians, have a third eyelid (the nicitating membrane) that helps them keeps their eyes moistened and allows them to better visualize at high speeds (or, for amphibians, underwater). The glands in birds' eyes allow them to secrete a fluid that is more resistant to evaporation than tears. The membrane also acts as a 'windshield wiper,' with each blink a birds' eyes are wiped free of excess fluid and debris.


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Are restriction enzymes active at −20 °C?
I have digested my DNA with NotI enzyme and put it in the −20 °C freezer without heat inactivating it. Can restriction enzymes work at −20 °C? Should I expect STAR activity?

I'd still recommend inactivating the enzyme or cleaning up the reaction after thawing up.

what is star activity? pls avoid acronms amap thnk y othrws t bcms unintellgbl

@AliceD Star activity is a pretty common term. It refers to nonspecific digestion by restriction enzymes.

@AliceD Star activity is not an acronym, it is merely restriction enzyme jargon. -edit- ninjad.

the capitals made it look like an acronym :) Thanks guys

Enzymes have temperature optima based on the organism they were isolated from. So I would predict the there is virtually no activity at −20 °C. Another consideration is that the reactions are likely to be frozen solid, so that would limit diffusion, and also slow it down. But the real question is: what are you afraid of? Just the star activity? Even if there were star activity, would that adversely affect your experiments?

I do not know, I have never had star activity before. Your answer makes sense and I think like that too but I posted the question here because there was no answer to this on the internet and I thought maybe someone here has an experience on this.

How about this paper? http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2396408/pdf/gkn182.pdf

@mdperry You should update that into your answer and perhaps include some excerpts from it, it is a great addition.

While there is unlikely to be any activity when the enzyme is frozen, there are better methods to prevent star activity.
1: If you are running the reaction in a PCR machine, you can program the machine prior to the digest to heat up to 65 degrees for 10 minutes after the intended digestion timeframe to heat inactivate NotI. Since NotI is a heat-labile enzyme, this prevents star activity by denaturing it.
2: You can use NotI-HF, which has been engineered by NEB to greatly reduce the amount of star activity and can therefore be left incubating at 37°C overnight.
3: You can gel purify or perform a "PCR purification" on your digested DNA samples, this also removes any protein present and therefore prevents star activity.

@WYSIWYG Why the scare quotes, though?

Additionally you can run the reaction in PCR tubes in the thermocycler. Simply choose the temperature you want to have it and then ramp up to 10 min at 65°C afterwards. No extra work needed and very convenient.

@Chris That was exactly what I meant. Sorry for being unclear. Edited answer.

@Chris WYSIWYG edited the answer to "PCR purification" (you can check the log). I was unsure why this was formatted as such.

Ah, now. I usually run these reactions in PCR tubes in the thermocycler, because the volume is small anyway and it is very convenient.

@MarchHo the quotes because it is not really a standard name of the procedure I guess. This term is popularized by Qiagen :P   You can remove it if you want.


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Can molecular genetics make a boolean variable from a continuous variable?
In the same kind of idea than this question. Gene expression are regulated through complex interactions. The concentration of enhancers and repressors is an important aspect that dictate the level of expression of a given gene. These concentrations can take different value on a continuous scale.
Imagine a case where fitness is maximized when a given gene produce n proteins per minute if the concentration of a given protein is greater than x. If the concentration of the protein was to be lower than x, then the gene should not be expressed (0 proteins per minute are produced). In such case, it would be great if a bunch of reactants were to be able to simulate a "switch function" that would switch from "NO EXPRESSION" to "EXPRESSION" at x.
It seems to me that such switch function should be very complicated to evolve. I would suspect that all chemical reactions, including the binding of enhancer to promoter region should follow the law of Michaelis-Menten and the Michaelis-Menten function is not at all switch function. So, I have been thinking about cooperative binding. Hill's equation describes a function that is effectively a switch function given that the hill coefficient is high enough. However, seeking a bit in the literature, it's seems that the Hill coefficient never really overpass 3 (or 5 for extreme estimates). A Hill coefficient of within this range gives a logistic function but still looks quite suboptimal compared to what a perfect switch function could do.
Are there switch functions in molecular genetics that could translate a concentration into a TRUE/FALSE signal?
How well do they simulate the perfect switch function?
Are they based on cooperative binding or on some other mechanism?

References for estimate of Hill coefficients:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1435008/pdf/biophysj00200-0109.pdf
http://onlinelibrary.wiley.com/doi/10.1046/j.1537-2995.1983.23283172857.x/abstract
http://www.pnas.org/content/93/19/10078.short
http://www.ncbi.nlm.nih.gov/pubmed/8816754
http://www.fasebj.org/content/11/11/835.short
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2816740/

Positive feedbacks can be one alternative. Positive feedbacks exhibit bistability and can therefore adopt one of the two stable states depending on the initial condition. A famous example of a positive feedback switch would be that between cI and Cro in λ-phage, which repress each other. Positive feedbacks also display hysteresis: if the state of the system is dependent on an inducing ligand X such that system attains state S2 at high levels of the ligand, then to switch from S2 to S1 the concentration of X would have to be reduced below the level that was required for the S1 to S2 switch; and vice-versa. In other words, the system tries to remain in its current state.
If the transcription factors that constitute the genetic positive feedback have a co-operative behaviour then you can expect a steep switching curve.
For details see this review.

Transcription interference can also be a good mechanism to provide such switches. Transcription interference occurs between genes in close adjacency to each other. This is basically, as the term suggest, that the neighbouring genes can interfere with each others transcription (by overlapping promoters blocking transcription initiation, collision of transcription machinery on 3' ends etc). In this article all of this is well explained along with a theoretical model called Transcription Interference network.
So a switch mentioned in the question can be carried out by this mechanism. Let's take two genes (A and B) that exhibit this transcrigption interference phenomena. Also let's imagine a gene orientation where the tow genes face each other with their 3' ends like in the picture below ( part B3 but with smaller overlap at the 3' ends). The setup is that gene A is 'stronger' than gene B ( has higher transcription initiation rate), thus at the 3' prime ends it can suppress gene by knockin off the polymerase (there are many possible scenarios when two RNAP-s collide or meet one knocking off the other is just one of them). Now if gene A's activity were to be lowered then gene B could over take and nullify gene A with the same mechanism seen before. In this case transcription initiation would still occur on gene A but it could not produce viable RNA because transcription would terminate early. This results in complete shut down in gene A's expression.

(source the linked article above)
Since gene expression and transcription are 'leaky', some minimal activity is basically always present, the above mentioned mechanism can be taken care of. In the article I linked there are many other examples how such switch could be carried out by transcription interference I just took one of them. Also you'll notice that the Transcription Interference Network model is based on such switches.


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Are there sterilisation methods that do not denature proteins as heat does?
Context:
Most countries require milk to be sterilised through radiation or heat to remove possible harmful bacteria. Both of these processes denature the proteins in the milk (ref).
Are there methods of sterilisation that will not denature proteins?

does radiation denature protein?

Why would a denatured protein in milk cause problems in the human body? Could you provide source / example for this?

About what problems in the human body are you talking? Please provide a source for this claim.

Read about pasteurization. Plus denaturing the native proteins won't cause any problem unless of course it coagulates to form an insoluble mass; in any case it is going to be digested in the stomach

I'd recommend removing the claim about human health as it's very questionable. Sterilisation by heat can, depending on the actual temperature, indeed denature proteins. Similarly, strong radiation will ultimately break molecules. I can't tell you whether the levels used to sterilise milk are sufficient to denature anything contained in it though. In general of course, "Are there sterilisation methods that do not denature proteins as heat does?" should be a valid question.

Filtration through a 0.45 micron filter is how we used to routinely sterilize laboratory solutions that we could not sterilize in the autoclave (or if we were in a hurry). This works primarily based on molecular size, so, for example, the viruses, bacteria, and fungi that are larger than the pores  will be retained on the filter, and everything else in the solution will pass through. There are filters available with different sized pores. I have never tried this with a colloid like milk. It seems like this Question was placed "On Hold" so I don't think I could "Answer" it, hence a comment.

Most countries require pasteurization and not sterilization of milk (https://en.wikipedia.org/wiki/Pasteurization), but that also involves heat. It doesn't result in denaturation of the proteins however - who would drink curdled milk?


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Blast output - program to count the number of hits
I have to use the old version of Blast (not Blast+) to compare DNA sequences and I have to know the number of all hits. They are not given in the header and I have to know them for many many of blast outputs and its nearly impossible to count them (or to export them in excel). I think I probably have to write a 'Perl Script' and I did my best but I am not familiar with programming and it is not working. Furthermore I had a look on the internet and they are always comparing it to BIO Search. I have e code, which for me looks pretty good,but it also done a comparison with BIO Search, what I don't need/want.
use strict;
use warnings;
use Bio::SearchIO;

Usage information
die "Usage: $0 <BLAST-report-file> <number-of-top-hits> <output-file>\n", if (@ARGV != 3);

my ($infile,$numHits,$outfile) = @ARGV;
    print "Parsing the BLAST result ...";
    my $in = Bio::SearchIO->new(-format => 'blast', -file => $infile);
    open (OUT,">$outfile") or die "Cannot open $outfile: $!";

# print the header info for tab-deliminated columns
print OUT "query_name\tquery_length\taccession_number\tlength\tdescription\tE value\tbit score\tframe\tquery_start\t";
print OUT "query_end\thit_start\thit_end\tpositives\tidentical\n";

# extraction of information for each result recursively
while ( my $result = $in->next_result ) {
    # the name of the query sequence
    print OUT $result->query_name . "\t";

        # the length of the query sequence
        print OUT $result->query_length;

        # output "no hits found" if there is no hits
        if ( $result->num_hits == 0 ) {
      print OUT "\tNo hits found\n";
         } else {
      my $count = 0;

                # process each hit recursively
        while (my $hit = $result->next_hit) {
            print OUT "\t" if ($count > 0);
                            # get the accession numbers of the hits
       print OUT "\t" . $hit->accession . "\t";
                        # get the lengths of the hit sequences
                        print OUT $hit->length . "\t";
                            # get the description of the hit sequences
       print OUT $hit->description . "\t";
                        # get the E value of the hit
            print OUT $hit->significance . "\t";
                            #get the bit score of the hit
       print OUT $hit->bits . "\t";

                        my $hspcount = 0;

                        # process the top HSP for the top hit
            while (my $hsp = $hit->next_hsp) {
                            print OUT "\t\t\t\t\t\t\t", if ($hspcount > 0);
                             # get the frame of the query sequence
        print OUT $hsp->query->frame . "\t";
                                # get the start and the end of the query sequence in the alignment
                print OUT $hsp->start('query') . "\t" . $hsp->end('query'). "\t";
                                # get the start and the end of the hit sequence in the alignment
                print OUT $hsp->start('hit') . "\t" . $hsp->end('hit') . "\t";
                                # get the similarity value
                printf OUT "%.1f" , ($hsp->frac_conserved * 100);
        print OUT "%\t";
                                    # get the identity value
        printf OUT "%.1f" , ($hsp->frac_identical * 100);
                    print OUT "%\n";
                                $hspcount++;
                            }
       $count++;

                        # flow control for the number of hits needed
            last if ($count == $numHits);
        }
        }
}
close OUT;
print " DONE!!!\n";

May someone help me, please. I need a program, which first tells me the header and then the number of hits. I am a little bit desperate and would be so happy if someone can help me. That would be so nice. Greetings Tina

Could you provide more info on how it's not working? I mean does it run but prints wrong output, or it won't run at all?

If your issue is related to your perl script then this is not on topic in biology-se. If you are interested in general procedure to get total number of hits you can see the above link.

Can you paste the output format?

Thanks for the fast answer. The script i didn't write by myself, I just found it on the internet. I don't get any output, because it works with BioSearch (you have to download). But I think with little changes it could work...but i am not sure (4th line??). Maybe there is a easier way. I just wanted to ask here, if someone has an idea how it will work or how i can reach my aim, counting the hits. In the link you posted is written, that the hits are in the header, but there are not, in my case.

@Nina Paste the first 10 lines of your output. Without knowing how the output looks, we cannot help you

The scrip won't run because it has a missing package that it wants to use (use Bio::SearchIO;) so you either download it , or use a different script

What kind of output do you have? I would recommend the XML output, because it contains all the information and not only a part like the table output for example.  Use -outfmt 5 as parameter. Example: blast -db dbname -query path/to/query -out path/to/outfile -outfmt 5

@Nina The script you posted from the Internet has an absolute dependency on the Bio::SearchIO module of the BioPerl library. The script uses Perl's object-oriented syntax to parse the blast search output file that you specify on the command line. There are a few books that may be of help. Ian Korf and Mark Yandell have a book named Blast from O'Reilly, with working examples of Perl scripts for parsing blast reports. Jim Tisdall has two O'Reilly books, Beginning Perl for Bioinformatics AND Mastering Perl for Bioinfo. One of them has a chapter on parsing blast output (as I recall)

This script, named blast2table.pl is from the code accompanying the Blast book by Korf & Yandell (there is a 3rd author whom I haven't met).
#!/usr/bin/perl -w
use strict;
use Getopt::Std;
use vars qw($opt_p $opt_b $opt_e $opt_m $opt_n);
    getopts('p:b:e:m:n:');
    my $PERCENT = $opt_p ? $opt_p : 0;
my $BITS    = $opt_b ? $opt_b : 0;
    my $EXPECT  = $opt_e ? $opt_e : 1e30;
my $START   = $opt_m ? $opt_m : 0;
    my $END     = $opt_n ? $opt_n : 1e30;

my ($Query, $Sbjct);
my $HSP = "";
    while (<>) {
        if    (/^Query=\s+(\S+)/) {outputHSP(); $Query = $1}
        elsif (/^>(\S+)/)         {outputHSP(); $Sbjct = $1}
        elsif (/^ Score = /) {
            outputHSP();
            my @stat = ($_);
            while (<>) {
                last unless /\S/;
                push @stat, $_
            }
            my $stats = join("", @stat);
            my ($bits) = $stats =~ /(\d\S+) bits/;
            my ($expect) = $stats =~ /Expect\S* = ([\d\.\+\-e]+)/;
       $expect = "1$expect" if $expect =~ /^e/;
            my ($match, $total, $percent) = $stats =~ /Identities = (\d+)\/(\d+) \((\d+)%\)/;
            my $mismatch = $total - $match;

        $HSP = {bits => $bits,
                    expect => $expect,
                    mismatch => $mismatch,
                    percent => $percent,
                    q_begin => 0,
                    q_end => 0,
                    q_align => 0,
                    s_begin => 0,
                    s_end => 0,
                    s_align => ""};
        }
        elsif (/^Query:\s+(\d+)\s+(\S+)\s+(\d+)/) {
            $HSP->{q_begin}  = $1 unless $HSP->{q_begin};
            $HSP->{q_end}    = $3;
            $HSP->{q_align} .= $2;
        }
        elsif (/^Sbjct:\s+(\d+)\s+(\S+)\s+(\d+)/) {
            $HSP->{s_begin}  = $1 unless $HSP->{s_begin};
            $HSP->{s_end}    = $3;
            $HSP->{s_align} .= $2;
    }
}
outputHSP();

sub outputHSP {
    return unless $HSP;
        return if $HSP->{percent}  < $PERCENT;
        return if $HSP->{bits}     < $BITS;
        return if $HSP->{expect}   > $EXPECT;
        return if ($HSP->{q_begin} < $START or $HSP->{q_end} < $START);
        return if ($HSP->{q_begin} > $END   or $HSP->{q_end} > $END);
        foreach my $field ('percent', 'q_align', 'mismatch', 's_align', 'q_begin', 'q_end', 's_begin', 's_end', 'expect', 'bits'){
        print "$field not defined\n" if not defined $HSP->{$field};
        }
        print join("\t", $Query, $Sbjct, $HSP->{percent}, length($HSP->{q_align}), $HSP->{mismatch}, countGaps($HSP->{q_align}) + countGaps($HSP->{s_align}), $HSP->{q_begin}, $HSP->{q_end}, $HSP->{s_begin}, $HSP->{s_end}, $HSP->{expect}, $HSP->{bits}), "\n";
    $HSP = "";
}

sub countGaps {
    my ($string) = @_;
        my $count = 0;
    while ($string =~ /\-+/g) {$count++}
        return $count;
}


text:
Can someone who cannot talk still whistle?
Can someone who has a damaged larynx, which does not allow them to talk, still create a tune when they whistle?
I know that the larynx is what allows a person to manipulate their pitch and volume, but whistling sounds are just air making a noise as they are through the lips.
Would a person with a damaged larynx be able to whistle physically, but it would just be a toneless noise?
If this is so, is there any situation where a human would not be able to talk, but could still whistle with a tune and understand the speech of others perfectly? (i.e. having not suffered brain damage or are deaf)

Whistles, trees and other objects can whistle with a wide range of frequencies...

This is an entirely new question. Perhaps you could roll back these edits, restore the original question and pose this question as a new one. Changing a question entirely into a new question is not recommended, the.more since it has been answered already,  this answer has been upvoted and quite extensively edited.

Sounds such as certain phonemes are made merely with the use of air flow and movement of the lips or tongue, without the need for action of the vocal chords. Think of 'f', 's', 't', k in the English language. These are called unvoiced phonemes in phoniatrics (see the phonemic chart). Also, clicking sounds and whistling can be made without use of the vocal chords.

Could you provide some references that this really works?

I sometimes fell the same, yet what I think simple basic stuff for me might not be for someone else.. also for me pronouncing letter b does resonate my vocal cords unlike the other letters you listed. In the link you provided B is listed as Voiced Consonants..... it's a bit controversal I think.

@poka.nandor - auch, the 'b' was incorrect (only without the 'e' it is unvoiced), thanks! I added a ref to a chart. Thanks. Is this OK? Mostly I seek journal papers, but this is so basic that it is textbook stuff and I don't have any phoniatric stuff around I'm afraid. I used to work at an ENT department, that's why it was so natural for me to answer without backup :) Thanks for the downvote, that was necessary (and for the undownvote :-).

Thx for the update :) I think by the way that voiced and unvoiced phonemes might be a little language specific.

@poka.nandor - yes! Next edit underway :)

Yes, as long as the person is able to blow air and 'shape' it using their tongue+lips, a person can whistle; even if it means this person lacks the ability to create the vibrational patterns necessary to talk.


text:
Why can't the human eye focus blue light?
I recently noticed that it is hard to focus on blue light sources, especially at night. When observing a blue light source, e.g. a neon sign, it looks somewhat blurry. A sign with a different colour right beside it looks sharp.
I already know about the three kinds of cone cells in the human eye (I'm not a biologist) with their spectral sensitivity peaks in in short (S, 420–440 nm), middle (M, 530–540 nm), and long (L, 560–580 nm) light wavelengths [1]. But does the spectral sensitivity correlate with focus? Or does our eye lens refract blue light in a different way?
When I screw up my eyes looking at a blue light, it becomes less blurry, but then all the other colours are blurred.
[1] http://en.wikipedia.org/wiki/Cone_cell#/media/File:Cones_SMJ2_E.svg

Interesting observation. In addition to aberration, which I suspect to play very little part, I'd hypothesis the role of neural network in visual pathway. What happens when only blue light detectors fires in this network?
Additionally, blue light detectors usually have 1/3 of sensitivity than other colors detectors. Also if a blue-detector is tightly connected with other blue-detectors (I don't know if this is true) then exciting one with also excite few others thereby giving a blurry image.

Shouldn't visual lateral inhibition reduce this effect and therefore sharpen the image?

It might, then the suggestion would be that we dont have strong lateral inhibition among blue detector neurons. just a hypothesis.

The same thing happens in photography when you see an image with colorful shadows of objects on it - this is called chromatic aberration. Check this wiki page if you're not familiar with. Now this happens because the lens in the objective has different refractive properties for different wavelengths or if go the other way around different wavelengths refract differently within the same material. This is why the famous triangular rainbow prism is capable of separating the sunlight to rainbow. So back to the question: in our eye there is the eye lens that is responsible for focusing. Let's assume that it is homogenous enough. So when light enters into our lens it refracts to separate colours and our lens is not complex enough to fully compensate for this. The second picture is a good approximation of the human eye lens in shape.
Edit: so when you focus one color perfectly the others will be blurry a bit since those will be out of focus due to different refraction.

taken from: http://commons.wikimedia.org/wiki/File:Prism-rainbow-black.svg

taken from:http://en.wikipedia.org/wiki/Chromatic_aberration#/media/File:Chromatic_aberration_lens_diagram.svg

This does not answer the question fully, as it only explains why blue is out of focus when the eye is focused on an object of lower-frequency (eg red). OP tried to focus on both.

umm well yes, but I thought that it's obvious that the logic goes vica-versa, yet this is an assumption that my logic works for everyone else.... :) Answer edit here we go!

Combination of factors:

Red and green (which are, from the evolutionary standpoint, the same frequency, as the red and green cones are recent variants of one another while the blue cones are distinct, they use a different photoreactive enzyme) are the dominant resolution source for human vision, while blue cones are much more important for expanding our ability to detect color (chrominance). It's why we have more red and green cones than blue ones (IIRC the ration is 10:1, though that may be way off).

Since focus is much more important to spatial resolution (details) rather than color resolution, the human "autofocus" is biased to favor the red-green band. Blue gets short shrift because of its relatively minimal importance to resolution. An experiment: if you mess around with the three color channels of an image in a photo program like Photoshop, messing up the blue channel affects the final color image the least, given the same amount of "messing" (like noise or deresolution).
Last but not least: as noted, chromatic aberration means that the different colors won't quite match up. It's not normally visible, likely because our brains auto-compensate when combining the different cone inputs into the final image (Canon digital cameras recently acquired a similar ability.) Not everyone is going to notice the effect: this is probably because blue focusses "shorter" in terms of lens focal length than red. So, you are much more likely to notice "blue fringing" if your unaided vision tends towards the nearsighted side (like mine does), given the focus center on red/green -- and you'll see it worst with distant blue sources than near ones. If you know someone who has worse nearsighted vision than yours and wears glasses (and neither of you have astigmatism) try using their glasses and see what happens.

Welcome to BiologySE... thanks for your contribution. Could you please add some references to back up your claims? We generally appreciate all answers and questions to reference outside sources (and not just personal knowledge). You can just edit your answer and add references either in the text or at the bottom. Thanks!

Light is scattered by gas molecules in air. The shorter the wavelength of light, the more it is scattered by the atmosphere. Because it has a shorter wavelength, blue light is scattered ten times more than red light. One reason is that
blue light has a frequency that is closer to the resonant frequency of atoms than e.g. red light (Exploratorum). Due to this scattering the eye has difficulty focussing as object edges get fuzzy.
The scattering of blue light degrades vision in normal day light, especially in foggy conditions when light scattering is at its worst due to the droplets of water in the air. Yellow filters (for example yellow sports sunglasses) improve visual acuity by filtering out the aberrant blue light (Laramy-K Optical).
Fun fact
The predominant scattering of blue light makes that the sun is clear in the sky as a yellowish circle, while the sky is blue due to the fact that it is the blue light predominantly being scattered away from the sunlight.

As well as natural shortsightedness to blue, due to chromatic aberration in the lens of the eye, there are also no S cones at the centre of the fovea. This means that focussing on a point source of blue is difficult and can be uncomfortable for some observers. Hope this helps.


text:
Why can we use mouse-produced antibodies on mice tissues?
I have seen biologists use mouse grown primary antibodies in mouse tissue, and they've told me that if the blood is perfused well then there is no problem with this method. How does the secondary antibody (anti mouse) not stick everywhere on all the mouse antigens in the tissue?
Clarification: I  understand that the secondary antibody is very specific to all mouse "class" primary antibodies. Can I ask then why using a secondary mouse antibody does not recognize background antibodies in the tissue, the ones that the mouse produced for itself naturally before I added any of my primary antibody? Are the existing primary antibody levels already present in the mouse much lower than the levels of the primary antibody I am adding?

The reason why the secondary antibody only recognizes the primary antibody is relatively simple: It is directed against the Fc-part of the primary antibody. As this part is species specific (but constant between all antibodies from one species, hence the name constant) a secondary anti-mouse antibody is directed against all mouse antibodies.
The general principle can be seen in the figure (although this is from immunolabelling, the principle stays the same, Image from the Wikipedia):

The anti-mouse secondary does recognize the antibodies which are present in the tissue. These are usually present at low levels (unless you work with immune tissues as spleens) and only cause a relatively low background. If it causes a problem, you can treat your sample before you add the primary with an Fc block which blocks the antibodies present in the tissue so that the secondary antibody will not recognize anymore later.

I think we essentially said the same.

Antibodies are very specific on what they recognize (this is called epitope that is the small part of the antigen that is recognized by a specific antibody), and secondary antibodies are made to attach to a specific part of the primary antibody - you can achieve this with monoclonal ABs. Antigens can be recognized by many antibodies (because they can have many epitopes for different antibodies), but usually a single antibody recognizes only a few or more likely a single antigen at max (since the specific epitope it attaches to is only present on a few or a single antigen(s)).
The wiki page for antibodies has plenty of detail on how ABs work.


text:
Why do mitochondria fuse together?
Contrary to all of the textbook images of mitochondria that I have seen over the years, I had just learned that the mitochondria within a cell form a dynamic branching network along microtubule scaffolding by fusing with each other.
Why would they need to fuse together?
When they fuse, would this involve only the outer mitochondrial membrane?
Would it not behoove a mitochondrion to remain distinct from others both physically and genetically?

Mitochondria produce a large quantity of reactive oxygen species (ROS) that damage the mitochondrial genetic material. These cell organelles do have a  DNA repair mechanism similar to that of the nucleus to some extent 3 which is not entirely understood however they have multiple copies and so the fusion and fission of mitochondria work together to maintain as many viable/healthy mitochondria as possible.
When two mitochondria fuse, their genetic material can recombine and thus exchange their damaged DNA. In this way, one of them repairs its DNA at the expense of the other accumulating more damaged DNA. Mitochondria that accumulate too much damaged DNA will 'die' - get eliminated, but they can be replaced by fission of healthy ones. Take a look at this wiki page, it has useful info.
Edit: This article may also be useful. (Mitochondrial fusion and division: regulation and role in cell viability
Giovanni BENARD and Mariusz KARBOWSKI, Semin Cell Dev Biol. 2009 May;20(3):365-74.)

Mitochondria are granular or filamentous organelle, which are present in the cytoplasm of a cell. They are ovoid in shape, with the presence of double membranes.
Mitochondrial fusion is required to distribute mitochondrial DNA to the mitochondrial population and to maintain its respiratory competent and energized organelles. Mitochondrial division is required to efficiently distribute organelles to distal parts of cells.

Can you please add some references to your answer?


text:
Does mung bean nuclease cleave a phosphate group when it's chewing off 5' or 3' ssDNA ends?
I'm looking to create blunt ends from sticky ends with mung bean nuclease for subsequent ligation.  Does anyone know full mechanism by which mung bean nuclease will do this?
In particular after the reaction, will the ends of resulting dsDNA have phosphate groups or will I need to phosphorylate the ends with an extra step for successful ligation?

According to Kroeker WD, Kowalski W, Laskowski M. 1976. Mung Bean Nuclease I. Terminally Directed Hydrolysis of Native DNA. Biochemistry 15(20):4463-4467

It was concluded that the products of the terminally directed hydrolysis of native DNA possess 5’-phosphoryl groups because: (1) greater than 99% of acid-soluble activity applied to the column was recovered in products which coeluted with 5’-terminated standards and (2) the w-monophosphatase activity of mung bean nuclease would have dephosphorylated 3’-phosphoryl-terminated products. The endonucleolytic activity of mung bean nuclease on denatured DNA also results in the production of 5’-phosphoryl-terminated products.

Also, from Promega:

Mung Bean Nuclease catalyzes the degradation of single-stranded DNA and RNA endonucleolytically to yield 5´-phosphoryl-terminated products


text:
Similarity between a heart attack and a spasm
When someone is having a heart attack, could it be considered , in some situations, a spasm? Below, I have written how I believe the process may work.
The heart is basically a muscle working constantly to create a sinus rhythm. As I understand it, one can get muscle spasms in any muscle (correct me if I'm wrong). If so, then couldn't someone get a muscle spasm in a part of the heart muscle that disrupts the sinus rhythm and causes chaotic signals? Thus causing a spasm throughout the muscle.
When one is having a heart attack, does the electric shock used cause a dramatic and fast 'stretching' of the heart muscles allowing the heart to get over the spasm and back to normal rhythm? Could something else, like an ElectroMagnetic Pulse be used to 'stretch' the heart muscles away from the spasms?

Do you think you could elaborate a bit here? Are asking what kind of damage could be done by this happening? From what I understand, a dramatically fast stretching of the heart could do serious damage to it; but I'll have to read a little more.

Doesn't the electric shock used to stop a heart attack cause dramatic fast stretching of the heart muscles? I'm basically asking is a heart attack like a muscle spasm in the heart muscles?

Okay. I understand! I'm going to run a couple edits on your question and you can see what you think of them. You can change anything back that you don't like.

You should be able to find either a review button or some such that will let you see my edits before someone else approves them.

I was able to do some reading and research and I found some interesting information that was a). news to me. And b). the answer is yes... And no.
A spasm can be the cause of a heart attack by causing the coronary artery to contract. When that artery contracts, it causes a dramatic decrease in blood flow to the heart. When the heart looses access to oxygenated blood it can cause chest pain (angina) or even an MI (Myocardial Infarction a.k.a. a heart attack). This spasm is can be caused by exposure to extreme cold, smoking and the use of certain recreational drugs.
However, this type of spasm cannot be stopped with defibrillation. Treatment for these involves administering Nitroglycerin and then later treatment involving Statin drugs and such.
In the terms you are speaking of, I don't see that that would be quite accurate. The most common cause of sudden cardiac death is VF (Ventricular Fibrillation) which is essentially an uncontrollable quivering of the heart muscle. I've never heard this described as a spasm though. Google searches of spasm and heart spasm have all lead to definitions of the word spasm and the coronary artery spasm mentioned above.
However, within that, I think you have a valid point and a decent understanding of how things work, just perhaps not fitting under the definition of a spasm.
When a defibrillator is used, it delivers a measured electrical shock to the person's heart and hopefully stops the chaotic electrical rhythm. I'm not sure if this part is correct however, I do not think you should look at that as a stretching of the muscle though. It is an electrical intervention that interrupts the former rhythm and hopefully the heart "forgets" what it was doing and returns to a sinus rhythm.
So, to summarize: A heart attack can be caused by a spasm; however, defibrillation does not help that type of attack. A defibrillator does not 'stretch' the heart but rather interrupts it's former rhythm. Because an irregular heart beat is not considered a spasm, I find it difficult to believe that any form of EMP would help it; however, I would love to hear that it has!
For more information on Ventricular Fibrillation, CAS (Coronary Artery Spasm) and AED's (Automated External Defibrillator(s)). Please see the following:
MedlinePlus - on V-fib
The Mayo Clinic - on CAS
HeartSine - on AED's
I hope this was helpful for you!
Edits:
In the comments, we had discussed a variety of possible causes and treatments for spasms, as well as what - if any - connection there is between how a spasm in perhaps a leg would be caused as compared to how one in your heart could be caused.
From the reading and research I did, I was fairly inconclusive about the safety or desirability of a device to prevent heart attacks using an EMP device implanted in your brain or on your brain stem.
As far as how similar a spasm in your heart is to a spasm in any other muscle, they are not very similar. As seen here, a muscle spasm in most parts of the body can be caused by numerous things, such as dehydration, poor blood circulation, mineral deficiencies etc. Whereas a heart spasm (know as a CAS) can be caused by alcohol withdrawal, stimulant use, extreme cold... As seen here.

Very useful info. I was wondering , when other muscles like a leg muscle has a spasm how are they treated? Could a 'tiny' defibrillator stop a leg muscle spasm?

Was it Galvani who experimented with a frog's leg and a small charge of electricity? Has anyone tried that experiment with an 'appropriate' electro- magnetic charge?

@201044 I did find somethings that discussed the use of an electrical pulse to control bladder spasms. Medicine.net.

The sinus rhythms (if that's what it's called)  that controls the heart pulses ; does this have 'part's' in the brain that regulate this rhythms. If so could electrical pulses effect these rhythms by effecting the associated parts in the brain. So if on had a heart attack maybe the 'relevant' parts in the brain or the spinal chord could be 'zapped' instead of the heart itself.

@201044 Yes, they are indeed called sinus rhythms. I will do some more reading and see what I find.

@201044 I did some additional reading on this and it looks like it may or may not be helpful but isn't necessarily dangerous either. (Although in someone suffering a heart attack it could be dangerous, the heart rate slows and then the person's blood pressure goes up). Here are two links you may find helpful, Johns Hopkins Anatomy of the Brain and PubMed.gov.

But maybe something similar to a pace maker could detect the precursors to a heart attack and if connected to relevant parts of the brain and / or spinal chord it could 'zap' them with tiny electric stimuli to prevent any subsequent attacks.

@201044 I'm afraid this has gone beyond my scope of understanding. I will tell you that not all heart attacks are caused by an abnormal rhythm; some can be caused by lack of oxygen due to a blockage or some such. Also, I think that part of the reason this has not previously been done is that it is much safer and easier to implant an AICD (Automated Internal Cardioverter Defibrillator) or pacemaker. Both of which can be implanted in an outpatient procedure. I hope to edit my answer later today.

Could an implanted device 'attached' to a part of the spinal chord and/or the brain moniter any known precursors to having a heart attack?

@201044 I think perhaps theoretically that could be done. But I'm just not sure how many people would ever be willing to line up to have the back of their head cut open to have a device implanted that could much more safely be implanted in their chest - near  their collar bone.

They might if the device prevented any precursors to any susceptibility to an attack.

@201044 I know at least two people who wouldn't. Myself and my mother... Just sayin' :)

What causes muscular spasms in any other muscle ,other than the muscles in the heart? Are the causes of a non-heart muscle spasm similar to what might cause a heart muscle spasm?

@201044 There are a variety of reasons, from diet to medications. I will share a link in my official answer with a list of common causes. The common causes of most muscle spasms are not at all the same as those causing a CAS (Coronary Artery Spasm).

What can cause a leg muscle spasm?

@201044 The same things that cause other spasms.

What is that, specifically that causes spasms?

@201044 I understand your question now! I'm just having a hard time finding information on it. I'll get back with you if/when I find something!

@201044 Perhaps that would be a good question to ask as a new question.


text:
Is there a biological neuron that spikes in response to hyperpolarizing currents?
As some background I've been building Electrophysiological models of neurons, and in the process stumbled upon a model, that in all respects is biologically plausible, but has a bizarre property I didn't think happened in nature. It spikes in response to hyper polarized currents.
My particular model has only one ion channel which is has similar properties to Hodgkin-Huxley's transient sodium channel. It has one activation gate and one inactivation gate.  The reason I only use a single Ion Channel was I was exploring If I could create a minimal spiking neuron with just a single channel. The answer is yes, but has different properties then I expected.
Basically when I inject a hyper-polarizing current it spikes and depolarizing current causes it to stop spiking.
I know that Depolarization block can occur, my current isn't that High, in fact its no current produces no spikes. It has a normal rest potential around -55 mV and Fires upward to about +40 mV.
Also note that If I change the parameters around I get a more normal neuron that spikes in response to depolarizing currents. So the property must be somehow related to the ion channel kinetics.
The question is: Are there biological neurons that spike when hyper-polarizing currents are injected?

This is a very interesting question and I hope it is well-received. Welcome to Biology S.E. if you have questions or need assistance, please visit The Help Center.

Could you tell us a bit more about the model? Does it follow Hodgkin&Huxley kinetics? Which types of channels are included in the model? Did you model a specific neuron?

@AliceD. I added some description of the model, hope that helps? I wasn't sure if it was allowed to post the entire equations that reproduce the equations, but I am more asking if the behavior is seen anywhere in nature.

Search for "rebound excitation" or "rebound spiking".

Short answer
Action potentials are always generated after a depolarization step.
Background
Action potentials are generated by prior depolarization of a neuron, typically by the action of an excitatory neurotransmitter. An action potential is per definition a sharp depolarization, followed by a somewhat slower re-polarization step. The most important step in an action potential is the activation of voltage-gated sodium channels (VGSCs). VGSCs open when the membrane potential exceeds a certain voltage upon depolarization (Liu et al. 2012), in turn initiating the depolarization step of the action potential. Hence, action potentials are always generated through depolarization.
The only ion channel I am aware of that may mediate a hyperpolarization-induced cation influx resembling that seen in VOSCs is the hyperpolarization-activated cyclic nucleotide-gated (HCN) class of channels. The associated current (Ih) is a non-specific cation flux (Na+, Ca2+). The HCN channel opens when the membrane potential hyperpolarizes. Thereby it generates a relatively small positive, depolarizing influx current. it is mainly involved in setting the frequency of pacemaker activity in, e.g., thalamic relay neurons that are involved in the oscillatory cortical activity characteristic of slow-wave sleep. However, Ih itself does not elicit spikes, it generates excitatory postsynaptic currents (EPSCs) associated with excitatory postsynaptic potentials (EPSPs), but not full blown spikes (McCormick & Pape, 1990).

Model evaluation and recommendations

Perhaps you identified EPSPs as action potentials? They are not.
Your resting membrane potential is very high (depolarized) - a more typical value is -70 mV.
VOSCs are opened at around -50 mV, hence your resting membrane potential is awfully close to action potential threshold. Any small perturbation leading to a bit cation influx will trigger action potentials. With a bit of stochastic leak currents included, a spontaneous spiking neuron is what you get. Ih (-like) activity may also set the stage for your out-of-the-ordinary observation.
Suggestion - lower your resting membrane potential to a more conservative value (-70 mV) and check the difference between EPSPs and action potentials. If you look at the following figure you can see that at your chosen resting membrane potential you are hovering between EPSP / actin potential threshold. A bi-stable situation results.

Action potential versus EPSP. Source: What when and how
References
Liu et al. BMC Neurosci 2012;25:2-9
McCormick & Pape. J Physiol 1990;431:291-318

Hey, did you check the chat room?

Thanks! It definitely helps. I got too caught up in the mathematics of the equations, and this helped me realize my parameters, where not in a biologically relevant range.

I was doing some more research and stumbled upon this paper here by Dr. Izhikevich that describe neurons as a system that can exhibit resonance. The author built a model that he calls the resonate and fire, which is a linear piece-wise model of a neuron.
The interesting thing, and why I bring it up, is it can respond to a "two inhibitory pulses, that if timed correctly will cause the neuron to fire. but neither inhibitory pulse alone can cause the neuron to fire.
Following some of the citations in the article (found here), apparently thalumus neurons can exhibit resonance and thus theoretically can fire in response to the right frequency of input. However it is important to note that they thalamic neurons are close to threshold, and are not receiving the double inhibitory input that Izhekivich talks about in his paper. The second paper uses a ZAP protocol.
A Zap protocol is injecting a Sine wave into a neuron and slowly change the frequency linearly in time.
$$I_{ZAP}(t) = \sin((\omega_0+\omega t) t) $$ where $\omega_0$ and $\omega_1$ are constants.

Not sure If I was allowed to answer my own question after accepting someone else's answer, but figured that this was relevant and useful to others.

You may answer your own question for sure! It is actually encouraged. However note that your question was on examples of real neurons and both cited papers are on models. +1 for your elaboration though :)


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Founders of Cnidarian colony
I know that colonial Cnidaria enlarge their colony by the means of asexual reproduction and sexual and asexual reproduction can originate individuals that will be part of other colonies.
Nevertheless, I cannot find any precise information about whether the individuals of a certain colony are all descendants of one founder or not. I thank you anybody for any answer!


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What does these $\Delta \Delta G$ numbers signify?
I was reading a paper and came across this table showing  $ \Delta \Delta G$ numbers of different nucleotide sequences in DNA/RNA. I know that $\Delta G$ is free energy and $ \Delta \Delta G$ is relative free energy with respect to L. variegatus 5 S RNA gene. What does this numbers signify actually?

Can you please link the paper. It is important to have the context of the measurement.

These are likely binding energies relative to a known interaction. Without seeing the paper, it's impossible to say for sure.

Very basically, what they've done in the paper was incubate different DNA sequences with histone octamers which were then separated on a gel. From this they could measure the ratio between nucleosomal and free DNA, which represents an equilibrium constant. This can then be expressed as a change in free energy ($\Delta G=-RTln(K)$), which is what the table shows. A lower (more negative) free energy change corresponds to a higher equilibrium constant which represents a greater proportion of DNA bound to histones. In other words, the free energy change quantifies the propensity of these different DNA sequences to form nucleosomes.


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What is an apomorphy? What is a plesiomorphy? Can someone confirm these definitions?
I want to know the correct definitions for the terms apomorphy and plesiomorphy. Unfortunately, there seems to be a variety of ways to define these terms depending on the language one uses. Thus, it has been hard to determine the correct definitions. I have settled on definitions that seem correct to me and also have examples. I would like confirmation from someone if I am understanding the definitions correctly.

Apomorphy -- any character state or trait novel to a species and its descendants. An apomorphy occurs when a taxon is selected to have a particular trait. Example: within the class reptilia, the suborder serpentes (snakes) has an apomorphy because its members have no legs.
Plesiomorphy -- an evolutionary trait or character state that is homologous within a particular taxon but is not unique to members of that group and therefore cannot be used as a diagnostic character for the group. Example: within the class reptilia, legs are a plesiomorphy for its members.

My Question:
Are these definitions and examples accurate?

That sounds pretty good. On this kind of things wikipaedia is very reliable.
You can also look at this very synthetic page:
https://www.enotes.com/homework-help/distinguish-between-synapomorphy-apomorphy-304128

The link has expired.

Apomorphy can simply be defined as derived trait or character state that is distinct to a particular species or group in a phylogenetic tree down to its descendants. The example you cited is fine.
Plesiomorphy is an ancestral or primitive traits that are homologous to a certain group of organisms but not unique to the other members of the group, hence cannot be used as a diagnostic character for the group.

Please try adding citation or scientific references to your answer.


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DNA-DNA cross-linking with formaldehyde?
The 3C (chromosome conformation capture) technology for studying chromatin 3D organization starts by a cross-linking step using formaldehyde to find segments of DNA interacting. In my understanding the cross-linking happens only between the DNA and proteins forming a DNA-protein-DNA complex. The "only" is from a DNA stand-point as of course protein-protein cross-links will also occur.
Can formaldehyde cross-link two double stranded DNA molecules? Is the 3C approach also capturing direct DNA-DNA interactions?
I looked at the literature, especially Dekker et al. description of the method, but couldn't find this information. Thanks.

Yes, it can: http://pubs.acs.org/doi/abs/10.1021/ja00074a005

@canadianer Thanks. Had a look at it and some papers they reference but apparently this is only true for single stranded DNA which is not the kind of DNA-DNA cross-link that might happen during 3C.

I've only skimmed the paper, but it constantly refers to interstrand cross links and cross linking of DNA duplexes.…

@canadianer Interstrand cross-linking refers to a cross-link between opposite DNA strands. Not really what I am looking for as in the case of the 3C it is two distinct dsDNA interacting.

years ago my lab mixed plasmid DNA and short polylysine peptides with terminal cystines so the peptides would polymerize on the DNA. Then they mixed in glutaraldehyde to crosslink the peptides. The resulting complex was stable in mouse liver weeks, but of course the DNA itself was useless.

Wouldn't direct double stranded DNA-DNA interactions in any substantial amount make DNA useless as a stable carrier of genetic information. This type of interaction could lead to inaccessibly deep foldings. Just my initial out of the box thought.

@vajra78 Not if those interactions are between two dsDNA regions (in cis or trans) and are not covalent (similar to protein-dsDNA or RNA-dsDNA bindings). Structure influences gene expression and the genomic function, this is actually the whole point of understanding the chromatin structure using 3C and related technologies.

I know structure influences gene expression and the genomic function. I was just thinking significant dsDNA-dsDNA interaction could lead to prion like knots because these types of interaction would be much denser structures.

@vajra78 sorry I actually missed your "substantial amount" point. Interesting, I don't know enough about prions to really judge about how likely would direct DNA-DNA interactions provoke them but nevertheless thanks, this was something I never thought about!

Actually plasmids form super coils, but there isn't covalent bonds formed as far as I know between dsDNA strands. I really shouldn't have jumped in this thread.

@vajra78 actually I am happy you did. That prion story was actually quite interesting. I will keep that in mind.

The fact that there is no inter-strand cross-linking between different double strands might be just because the cross-linker cannot bridge the distance between amines of different bases on different double strands.
The formaldehyde based linking of nucleobases has been described by Chaw et al. (1980) where formaldehyde bridged a gap of app. 3 angstroms (2x 1.3 A for N-C bond).
Even if you take the most condensed form of DNA as in nucleosomes (see X-ray model of nucleosome with bound DNA) you still have at least 20 angstroms between the bases of different double strands.
At the same time there are only 3 angstroms between the nucleobases of the same double strand, agreeing well with the length of a methyl-bridge.


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Should gene therapy safety protocol include isolation?
In the case of a gene therapy trial where viral vectors are used to deliver genes into mammalian cells, including humans, should biosafety and ethical protocols include isolation of the patient as a precaution post administration that the viral vector may recover its ability to cause disease and harm the environment (more so the patient as the animals tested can be destroyed by incineration)? And how long should that isolation stay be to ensure safe disarmament of the viral vector?

The viral vectors used in gene therapy trials are so heavily modified they can't just start replicating again. Chances are they are a lot more dangerous to the patient than they would be to anyone around them. I don't know what happens if you use a replication deficient lentiviral vector in an AIDS patient though.

But usually there is the delivery vector and the helper viral vector to help promote the new sequence, no?

@user137 you don't use lentiviral vectors in AIDS patients.

@Nederealm no, there's only one vector, delivered with a completely replication-deficient virus. Isolation of gene therapy patients is completely unnecessary.

@Nederealm It sounds like you're confusing the production of viral vectors with their application. The replication deficient viruses must be produced in cell lines that produce the missing proteins, and I think AAV only grows in cells that are also infected with adenovirus, though I don't know much about virus production.

So why is the argument of viral vectors gaining the ability to become a replication virus often brought up in an ethics debate?

I found my answer here > ISSUES RELATED TO PARTICULAR CLASSES OF VECTORS FOR GENE THERAPY http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/CellularandGeneTherapy/ucm072987.htm#vi


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Finding confidence level of miRNA disease associations
I'm an undergraduate computer engineering student, and I have a project about bioinformatics. In this manner, I need to find prediction( or association I'm not sure the correct terminology) confidence of miRNA disease relationships. Let me make more clear my question with an example. For instance, in one of the bioinformatics related databases( HMDD) it is said that, miRNA X is related with disease Y. I want to find that what is the confidence. I mean  you're 100% sure about such releationship? Do you know any database for that purpose? In HMDD I can only find miRNA - disease name couples it does not give any statistics.

I'm not familiar with the particular database, but isn't there any reference for such entry?

@poka.nandor Actually neither I am, after I asked this question, I looked at HMDD again and I saw that in the explanation of hmdd, it is said that "HMDD  is a database that curated experiment-supported evidence for human microRNA (miRNA) and disease associations." so, since it is curated experiment-supported I think they're 100% sure about their associations

I haven't come across with a reliable database. For the one that you are talking about, if they haven't provided any association 'scores' then you can download the entire data and calculate them yourself. Read the paper/additional data corresponding to the database; they must have done normalization and must have calculated some scores

You can start off by studying how an miRNA regulates the functioning of a gene. it basically binds to the 3'UTR and 5'UTR of an mRNA and restricts its translation. There will be genes associated with the disease of your interest,  find out the miRNA's related to those genes. You can use micro array data to find out the up-regulated genes. Target scan human, miRDB, are various databases for retrieving the miRNA's related with the genes.
The confidence level for finding out the association of certain miRNA's with the disease can never be 100%. but a relationship can be established by simply viewing the complementarity between the miRNA and the mRNA expressed during the diseased state.

Could you add links or citations for people to read further into this topic?


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Why are fruits so large compared to their seeds?
Why do many plants produce such large fruits(apples and strawberries,for example) if those contain only relatively small seeds?

Although I'm not an expert, after a bit searching, I've found this article : link , even if it does not mention about the seed-size relationship, it gives some information about the points that affect size of fruit

Much of the size of commercial fruits is due to selective breeding by humans.  Compare the size of a store-bought strawberry to a wild one, or an apple to a wild crabapple.  For wild fruit, the standard explanation is that the fruit serves as a lure: some critter eats the fruit, the seeds pass through its digestive system and are deposited (along with a nice bit of fertilizer) some distance away from the parent.

The short answer: Fruits are large compared to seeds because humans have made them large.
In the natural environment, there is a different set of evolutionary pressures. A fruit has to be able to successfully propagate itself using its seeds, while commercially farmed fruit is usually cloned via vegetative propagation. Therefore, the commercial farmed fruits do not need large seeds to propagate, since they are cloned by the farmers. In many cases, their seeds are actually nonviable (i.e. they will not grow when planted).
For example, this is a wild banana, before being selectively bred by humans. As you can see here, the seeds are enormous compared to those in commercial Cavendish bananas.

Similarly, this is a wild strawberry compared to its commercial farmed variant.

Fruits with large seeds don't appeal to consumers, and therefore farmers who sell fruits with large seeds will get a poorer return on investment.
Therefore, farmers who plant the fruits which appeal the best to consumers by being the easiest to eat (small seeds) will reap the greatest profits.

Not true about taste.  Farmers tend to plant what grows & ships well, and has eye appeal in the store.  Taste is way down the list of desirable factors, if it's considered at all.  Compare the taste of 'heirloom' varieties vs common commercial ones: for apples, a Cox's Orange Pippin vs Red Delicious or Honeycrisp.  Another factor is that commercial fruit is often treated with growth-enhancers such as gibberellins (or was, back when I was a farm worker), which adds watery size at the expense of flavor.

@jamesqf Fair point, updated.

@jamesqf Can confirm this, working with watermelons. Usually wilder varieties are smaller and more concentrated in flavor, but harder to eat due to seeds, etc.

Just to note, the Cavendish banana, like all other edible bananas doesn't just have small seeds, it is seedless. In fact, all banana plants (of the same variety of banana) are genetic clones of one another produced by vegetative reproduction rather than sexual reproduction.

@JackAidley "Seedless" in most cases just means their seeds are non-viable and reduced, not that they do not exist. New Scientist article

@MarchHo: Okay, fair point. What I mean is that it's not just that they have smaller seeds it's that these seeds are, as you say, totally non-viable.

Because seeds are not appealing to animals and they can only be carried by animals if they are made appealing - there are exceptions of course such as this one -. Seeds are covered with juicy layers that can be nutrient to animals in order to be made appealing and to be dispersed. Animals eat those juicy layers along with seeds, seeds resist digestion and fall on the ground where animals defecate. If the layer was thin and provided only protection to seeds, animals would not eat them and the seeds would have to be dispersed by other means. Therefore we would not call them fruits. The reason why seeds are not as large as their fruits is:

They have to be dry to resist climate changes while staying dormant below ground. And dry is generally small.
Seeds' task is to be dispersed, not to be nutritious to animals. And they usually do not taste very  well compared to fruits - some do taste well as we know but I do not know why -. So why make them large and irritate the animal? Make them small so they will be overlooked by the animal and be dispersed happily

Most fruit/seed evolution is probably a reflection of the plants propagation strategy and any existing symbiotic forager relationship.
The strawberry expands its range by creeping with feeder shoots. It is also heavily foraged by small birds , thus the exterior small seeds.
Nut trees i.e. walnuts , are on the other end of the spectrum. The seed is nearly the size of the "fruit". All they want is some little squirrel to shake them loose and fall to the ground, then try to outgrow everything else.
cherries....
Just making all this up.

If you are "just making all this up", do you mean it is nonsense? If yes, please remove the answer. It sounds OK though. If you support your answer, could you edit it to be a bit more professional? i.e. the "cherries..." doesn't make sense.

@AliceD: To a certain extent any answer that involves the 'why' of evolution has to be made up, because we're taking a situation that exists as a result of a multitude of factors acting over millions of years, and using reason to try to figure out the 'why'.  Since we weren't there watching every step, there has to be a lot of guesswork involved.

@jamesqf,  obviously, but while educated guesses are appreciable, nonsense, unfounded and  fabricated answers should be discouraged


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Amino acid compatibility
The (human) genetic code encodes 20 amino acids. They form a protein using peptide bonds. Each amino acid has a carboxyl group (COOH) and an amino group (NH2) that can potentially form a peptide bond.

Source: The Culture of Chemistry
Does this property of amino acids means that each amino acid can form a peptide bond in any combination? In other words, are there certain amino acid doublets in existence that never pair up?

Welcome to Biology S.E.! If you need additional assistance, please visit The Help Center.

Right, so any amino acids may be chained together. The limitation is that the function of a protein depends on which amino acids are in your polypeptide chain, and in what order. The cell mediates this on multiple levels: http://en.wikipedia.org/wiki/Protein

Exactly what is the question? Whether all amino acids are "compatible"?

I edited the question to prevent closure. I think it is a valid question with enough background. It was just formulated a bit unclear

Short answer: there are no restrictions in principle on which amino acids can follow which. That means that in principle you can have polypeptide in any configuration: AAAA, WQWQWQ etc.
Problem is that polypeptides must be functional and, because they are in aqueous solution, it puts restrictions on how polypeptide form secondary and tertiary structure. It means that having very rigid hydrophobic surface is energetically unfavorable. If cell where to produce such polypeptide, it might be either degraded or cause problems for cell, such as aggregates or other form of toxicity.
That means that polypeptides has more restrictions than just linkage (C-N bonding) order. And of course it has been studied: Probabilistic analysis of the frequencies of amino acid pairs within characterized protein sequences.
Authors analyzed protein databases to find out which doublets of amino acids are more and less common. First of all, not all amino acids (of which there are 20 considered) have same occurrence, e.g. P(L, leucine)=0.096, P(W, tryptophan)=0.0118.
And, of course, not doublets are equally distributed (16 pairs out of 400 were picked as statistically significant):

I think this is the better answer.

It seems like you may be thinking along the lines of DNA, where adenine pairs only with thymine and guanine with cytosine.
Unlike DNA, amino acid chains are single-stranded...
...so there is no pairing of compatible types like there is in DNA. Neighboring amino acids in the chain can be in any combination (as you say, because each has a carboxyl group and an amino acid group), just as neighboring nucleotides in DNA (ie those next to, not across from, each other) can come in any order.
In practical terms, the functional groups of amino acids have different chemical properties (hydrophobic, hydrophilic, aromatic), therefore some pairings are probably more commonly found than others. But in principle there is no restriction on who is next to who.

To answer your question: No, there are no restrictions to what amino acid is next ("a nearest neighbor") to its N-terminal or C-terminal neighbor.


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Oligonucleotide purification with desalting
I have ordered 36bp oligonucleotides that anneal to each other and create sticky ends to be cloned in a vector afterwards. I have tried cloning many times with different methods and I failed. Now I started to think that my oligos are actually shorter than 36 nt since desalting is not an effective method and most of my oligos do not create complete sticky ends when they anneal to each other. I believe I have also found support for this. So, how much is it possible that most of my oligos are actually shorter than 36 nt ? Is this a good troubleshooting or 36 nt oligos not being purified with more efficient method should not really create a problem?

I've never had much luck trying to clone something that small into a vector. One thing I prefer to do is to make the oligos long enough to have the restriction sites on each end, with 3 extra bases past those so the restriction enzyme binds better. I cut the oligos, then gel purify, then ligate. But still, it's a small insert. Something that small feels like you could almost PCR it in somehow.

Well, people do ligate annealed oligos directly to cut vectors. Actually it has been said that cloning small inserts is much easier than large inserts but I have no experience on this of course. If you are not familiar with oligonucleotide annealing for ligation and cloning, this may help you: https://www.addgene.org/plasmid-protocols/annealed-oligo-cloning/

Oligos from well-known companies are pretty good to my knowledge. Problems begin after 100-300bp oligos/dsDNA fragments.
What I suggest is PCRing your vector like that:
          Primer F: ---------------------__________
vector: ======|v site for 36 bp insert v|==========...
        Upstream                        Downstream
        ______----------------- : primer R

Legend:
__ portion of primer annealing to vector
-- portion that is your insert of interest

Then you can actually just transform that PCR into e. coli, which will seal two nicks together and propagate your vector. Treating your PCR reaction with Dpn enzyme will ensure that all template DNA is removed.


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How are Stem Cells Produced for Stem Cell Based Therapies?
I was doing an initial search on google but couldn't come up with anything I'm looking for. I know were stem cells come from and a bit about their levels of differentiation. For example, embryonic stem still becoming mesenchymal stem cells and what not. I also understand that epithelial tissue cells can be converted to a pluripotent stem cell state but at very low efficiency. So, all that aside how are stem cells obtained in the volumes necessary for stem cell based therapies without causing them to start differentiating during production?

Can you give a reference to a particular therapy you're interested in? Different treatments use different protocols.

I've heard of therapies, than in my opinion sound silly, were cancer patients are given a dose of stem cells intravenously. I would assume these would be made from an embryonic stem cell lines. If I'm correct, how are these lines maintained and generated? Is there a specific culture medium that can maintain the pluripotent state while still undergoing continuous growth?

Basically you culture them in a medium that inhibits cell differentiation. ES cells derived from Blastocysts are immortal (like cancer cells) as they express telomerase. Give the correct signaling molecules in the medium, they will continue to divide in an undifferentiated state, for all intents and purposes, indefinitely.

I didn't get your question exactly. If you wanna ask how to maintain the potential of stem cells, yes, there are several compound that could keep stem cells, such as Leukemia Inhibitory Factor (LIF). As far I know, the using of pluripotent stem cell, such as ES, iPS, still has the problem of formatting teratoma in vivo studying. It means totally differentiating pluripotent stem cell still need to be work out.

If you could explain what LIF is, then I think you have answered this question.

@AliceD LIF - Leukemia Inhibitory Factor. It is added to ES Medium to keep them from differentiating.

With LIF alone you also need to use feeders. Labs are starting to switch to 2i so that you only need to plate the ES cells. It makes DNA and RNA analysis much easier, because you do not need to try and remove feeders after the fact so you do not have the fibroblasts confounding your data.

@AMR - thanks for the explanation, and thanks for that edit too. +1

But I think there should be other compounds other than LIF. As far I know, some lab prefer culture human iPS cell without MEF (feeder layer), LIF and even serum. If you have an interest, there are lots well-known protocol in internet and papers. And also, lots of commercial mediums for raising stem cells, such as https://goo.gl/ImF1I8 .Following these compounds in mediums, you will know what can keep stem cell's potential.


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Can't resolve protein with native PAGE

This is a native gel. Let's call the left 2 lanes protein A and the right 2 lanes protein B. B is the same as A except it has a FLAG tag. They are both homotetramers of about 65 kDa. After purification, both appeared as single bands on SDS-PAGE. However, these are relatively old samples and I expect multiple bands due to degradation (which I've observed with previous samples). Either way, I cannot for the life of me resolve them with native PAGE. B is somewhat workable, though still atrocious. For each of A and B, I've loaded 5 and 15 uL (hence why there are two lanes for each) with 5 uL of 5X loading buffer. Yes, I know that's not the correct ratio; I can't imagine that being my problem but I'm running it again with the correct ratio. The rest of the conditions are pretty standard:

The protein was in PBS (50 mM dibasic sodium phosphate, 75 mM NaCl, pH~7.3) prior to adding loading buffer; I've also used TBS (50 mM Tris, 7.5 mM NaCl, pH~7.4) with similar results
4% stacking gel, pH=6.8
6% resolving, pH=8.8
Tris-glycine running buffer, pH~8.3, ran at 4°C with 23 mA constant current. I've also ran at 30 mA with similar results.
Stained with Coomassie

Let me know if any other info is needed. Any advice will be greatly appreciated.

@vajra78 Each sample is a pure protein.

what exactly do you mean with 'resolve'? "To get a clear band"? Why is B better (i.e., clear band) than A you think? Isn't A not just totally degraded?

@AliceD Yes, get a clear band. I have no idea why B is better than A, the only difference is the FLAG tag (ie greater charge:mass ratio). A is not totally degraded because I can resolve it with SDS-PAGE.

When I see a smear I suspect heterogeniety. Since the smear goes up, it suggests the proteins are heavier (though it's a native gel and you can't assume mass and migration are directly correlated). Is there some possibility that A is polymerized? And the FLAG tag on B somehow disrupts this polymer?

@user137 That's an interesting idea; I was thinking it was just a problem with my gel conditions. The protein should have no specific interactions with one another. However, each monomer has a free cysteine that could possibly form a disulfide bond with another monomer and link the tetramers together. The FLAG tag could just add some electrostatic repulsion to prevent close contact of the cysteines. It's kind of a nice explanation; however, I really don't expect it to happen readily. I'll add some mercaptoethanol to the loading buffer and see what happens.

@user137 Well thanks for the suggestion but unfortunately mercaptoethanol didn't fix the problem and I can't think of any other reason why these proteins would polymerize.