text:
How far in evolutionary distance is the P-distance estimator "accurate"?
I'm reading Yang's computational molecular evolution and in the very first chapter it says the P distance is a "simplistic distance measure" between two sequences. It is the number of different sites / the total number of sites.
However, because back mutation and multiple hits cause inaccuracy it become weak over longer distances (large number of generations to most recent common ancestor, MRCA, I guess).

"The raw proportion p is usable only for highly similar sequences,
  with p <0.05 (5%, approximately)."

How far back in divergence time do we have to go for this p<5% to be violated? I assume it has to do with the mutation rate right? I'm trying to get a sense of scale on this for myself. Are we talking a distance of just a few generations (within species), the distance between two closely related species, or an even greater distance?

Yes, obviously it depends on the mutation rate, which in turn depends on the locus you are dealing with and is organism-specific.


text:
How do you express and detect GLP-1 receptors?
I am currently working with a peptide which is an analogue for glp-1, but during invitro studies am not able to detect for the presence of GLP1- receptors.
The cell line used is Min-6. How do I detect it?

If you have an antibody against the GLP-1 receptor, you could make a whole cell lysate and do a western. How old are your cells? According to this they alter their behaviour over time and alter the expression of genes.

Try polyclonal antibodies. It might just be that your antibody isn't picking up the epitope.

does the cell line express GLP-1 mRNA? You could easily and cheaply use PCR to amplify the GLP-1 mRNA. That technique would be exquisitely sensitive and specific for what you want - and you don't need to worry about expensive antibodies.

The question is slightly unclear since it fails to clarify whether the GPL-1 receptor is endogenous or is over-expressed. Also does the GLP-1 belong the same species that Min-6 originates from or it belongs to a different species? Those are important points since if the receptor is endogenous then the main problem is sensitivity of the detection method, which can be difficult sometimes to detect an endogenous protein relative to an over-expressed one. Also if the GPL-1 belongs to a different species, which you are trying to over express in Min-6 it might not work since the cellular machinery might not support its production (tho unlikely) or its transport to the cell surface, which I have had this problem before. It could be that for the particular expression levels you have in those cells, if the GLP-1 is over expressed or if its non-endogenous, it causes toxicity and kills the cells that are expressing it hence why you do not see any GLP-1 expression (e.g due to excessive receptor activation). Also could you clarify what you mean by "analogue for glp-1"? does this mean it is not exactly GPL-1 or its a homologue or orthologue, which you are trying to detect using a GLP-1 specific antibody? that might again be a problem if the GLP-1 Ab you are using detects a region of the receptor, which is not conserved in its homologues.
In terms of solutions as pointed in the comments, the best way to go about this is to use a polyclonal Ab, which is raised against a large section of the peptide in question, allowing you a better chance of its detection. If that did not work, you might have to resort to GLP-1 mRNA detection using RT-PCR or qPCR, which you would need to design primers for based on you GPL-1 sequence which you can get help with here.

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text:
I don't wash my hands and never get sick. Any theories?
I have only been sick a few times in my life(I'm 21 and probably 3-4 times) and even when I am sick I don't feel that bad and I am over it within a day or two. My family has always given me slack for never washing my hands but I see no point unless they physically look dirty. Unlike me though my family gets sick around the same amount as other people(a couple times a year).
I have this theory that since my body is always exposed to germs and other bacteria that it is stronger and able to fight off most colds before they ever happen. This theory obviously contradicts popular opinion and what doctors recommend but I have always felt fine not washing.
What do you guys think?

it's well known that exposure to germs at a low level improves the immune system, but not washing your hands also increases the risk of exposure to doses of germs large enough to cause sickness.

You may very well be strengthening your immune system, but there are microorganisms that you don't want to get, which can have longer term health consequences. For example, there seems to be a significant increase in developing MS if you've ever had the Epstein-Barr virus. But this is just one case. You also don't want Salmonella, tape worms, etc.
So there may be a price to pay for a stronger immune system: namely higher probability of exposing yourself to the more dangerous things out there.

That is a minor fear of mine but the way I see it a lot of these diseases/infections are spread many other ways than through just being picked up on your hand. eg breathing them in/eating them etc. In which case a stronger immune system would be beneficial in fighting things off that may have nothing to do with washing your hands.

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text:
Dinuclei cellular mechanism
I am no biologist, but I have this question buzzing in my mind. It's a matter of curiosity
What happens when two nuclei occupy the same cell? Would we expect the embryo (if it lives) to have complex cells (assuming the nuclei are from different species, i.e., genetically incompatible with each other)?

It depends on the kind of organism.
When the cell with two nuclei will try to go through mitosis, the nuclear membranes will fragment, releasing all genetic material to the cytoplasm, where it will behave normally, as if it came from only one nucleus. The DNA will divide and move into two daughter cells, each of which will assemble its one nucleus from the membrane fragmnets. However, each daughter nucleus will have double the normal number of chromosomes - one whole set from each of the old nuclei.
(In fact, this is what happens in humans after fertilization - the male and female pronuclei never merge, they just copy their DNA and divide it simultaneously into two daughter cells. The pronuclei are haploid, so the resulting daughter cells are diploid.)
What happens later, to the tetraploidic cells from your question, when they try to develop into a full organism, may end better or worse.
For exapmple many plants will just grow normally, only form bigger plants (modern wheat is such an example - it may be tetra- or even hexaploidic, meaning it has up to 6 sets of chromosomes).
For animals this is usually fatal, but some, like the xenopus, seem to be unaffected.

the second sentence is very obscure. Due to this, it is absolutely not clear, how you relate binuclearity to polyploidy.

I hope it's better now.


text:
How can I fetch nucleotide sequences based on ENA accession numbers and positions?
I have a list of ENA accession numbers and a start and end position in each sequence. In the end I want to align all those sequences, but first I have to fetch them and the list is too long to do that manually.
Is there any existing tool or script that can fetch all those entries from a database and extract the correct subset of the sequence?

Could you show us a sample of the file? What's the format like? What operating system are you using? You will probably need to download each sequence and then extract the subsequence, all of which is easy enough but the details depend on how your information is formatted and the system you are using.

I will edit this if you update your question with more information, but for the moment I am assuming you have access to a Linux/Unix system and that your file of accessions looks like this:
A00145.1 123  456

Where the first column is the accession and the next two are the start and end of the region of interest. So, using the same basic approach as WYSIWIG's answer, you can combine this with fastasubseq from the exonerate suite of tools (on Debian based systems, which include Ubuntu and Mint, you can install by runnning sudo apt-get install exonerate). Just copy/paste this into your terminal:
while read acc start end; do
  wget -q -O "$acc".fa "http://www.ebi.ac.uk/ena/data/view/$acc&display=fasta" &&
  fastasubseq "$acc".fa $start $end > $acc.$start-$end.fa
  rm "$acc".fa
done < list.txt

With the example above, this command will create a file called A00145.1.123-456.fa which contains the subsequence of A00145.1 from positions 123 to 456. Since it first needs to download the entire sequence, it also creates the temporary file A00145.1.fa which is deleted by rm "$acc".fa. If you want to keep the file, just remove the rm line.
If you can't install exonerate, you can also extract the subsequence manually but that's a bit more complex:
while read acc start end; do
 wget -q -O "$acc".fa "http://www.ebi.ac.uk/ena/data/view/$acc&display=fasta" &&
awk -v st=$start -v end=$end '{
                               if($1~/>/){print}else{s=s$0}}END{print substr(s,st,end)
                          }'  $acc.fa |
 fold -w 60 > "$acc.$start-$end.fa" && rm "$acc".fa
done < list.txt

I think explaining these scriptlets in detail is not in scope here but I'd be happy to do so if you post a question over on unix & linux.

From ENA

To retrieve results by a single identifier for all records other than
  Taxon and Project please, use the following URL syntax:
http://www.ebi.ac.uk/ena/data/view/
For example, the following URL returns the EMBL-Bank entry with
  accession number A00145 in HTML format:
  http://www.ebi.ac.uk/ena/data/view/A00145

Check this.
If you use a *nix system (such as Linux, Unix, or OSX) then you can do this:
[ if list.txt contains all your ids ]
for i in `cat list.txt`; do wget -q -O $i".fa" "http://www.ebi.ac.uk/ena/data/view/"$i"&display=fasta"; done

OR ELSE
Download the entire database and pick up sequences based on your identifiers, offline. You can use your favourite programming language to retrieve sequences. [I prefer awk but python is also good]


text:
Why are transcripts translated from 5' to 3' direction?
What is the mechanism?
In other words, how does the tRNA know whether the codon is in 5' to 3' direction? I also heard that anything that is translated from 3' to 5' direction is degraded. Is it true?

Related: Why does DNA replication go from 5' to 3' http://biology.stackexchange.com/questions/477/why-is-dna-replication-performed-in-the-5-to-3-direction

The tRNA is not acting alone, it has the help of the Ribosome.

The Ribosome assembles at the beginning of the transcript and starts the translation at the first AUG codon. It then binds the first tRNA which fits to the mRNA. The tRNA is then moved from the A-position to the P-position and the next tRNA is binding (the move around and bind by chance. A nice animation is found in the Wikipedia article on translation (which is also the source for the image above).
Translation can't go into the other direction, it is always in 5' -> 3'.
To recognize the right direction (and the right starting point) the Ribisome is not simply starting at the 5`end of the mRNA. Before the start codon AUG the mRNA contains a regulatory, untranslated region, the so-called 5'UTR. The end of the 5'UTR of eukaryotes contains the so called methylguanosine cap (and is only present in mature mRNA), in proaryotes this is done by the Shine-Delgano sequence. Both are recognized by the ribosome. After binding to the mRNA, the ribosome slides along the nucleotide chain until it reaches the first AUG to initiate translation. The image is from this description (which contains a lot more details, if you are interested in it).

Thank you for your answer. But how does the Ribosome "know" that the beginning is THE beginning? What if sequence is flipped 180 degrees? if you look at the mRNA as a string of sequence, say AAAUUG, then the beginning can be either A or G, because if you flip the sequence, it's GUUAAA.

@Alby I edited and expanded my answer.

I think that there is no reason in principle why early evolution couldn't have landed on a translation mechanism going 3'>5'. There are, however, clear biochemical reasons why the transcript itself has to be made in a 5'>3' direction. So in this alternative world where the initiation signals would have been at the 3' end of the mRNA, the message would have to be fully transcribed before the anti-ribosome could bind to these initiation signals.
In bacteria at least, translation is initiated at the 5' end of the mRNA even as it is being synthesised, and this rapid transcription>translation link may offer some advantages.

I don't think this happens in eukaryotes, since the mRNA has to be processed and exported from the nucleus.

Agreed, but I'm assuming that the bacterial system is closer to early life.


text:
Why do people use anti-inflammatory drugs?
Why people should take anti-inflammatory drugs, when inflammation is a physiological way to defend our organism?

You are correct in that inflammation is a physiological process. It is vital for the correct development and localisation of a strong immune response, and contributes to tissue healing.
However, the beneficial effects are replaced by dangerous processes when they are prolonged or too intense. This includes both effects of leukocyte attraction and invasion, but also deleterious consequences of the accumulated cytokines. For example, TNF-alpha originally contributes to immune cell activation but in prolonged inflammation prevents tissue healing.
Note that "prolonged inflammation" does not necessarily mean that inflammation has been apparent or noticeable for the affected person for a long time. Often by the time symptoms present, the inflammation process has been persistent and become pathological. At this point, anti-inflammatory drugs need to be used as soon as possible to minimise irreversible damage.
Aside from this, quite often inflammatory and immune processes are triggered without a need for them. Our immune system is quite prone to errors, and cases of misguided immune reactions are actually increasing. Consider for example allergies, which often manifest with skin-symptoms such as eczema that can be treated with anti-inflammatory creams.

While inflammation is a natural defensive process, it also does (sometimes severe and/or permanent) damage to tissue. Therefore, when inflammation has been caused by an injury or an auto-immune disease such as rheumatoid arthritis, anti-inflammatory drugs do more good than harm.

What really bothers me is a situation when you take a simple anti-inflamatory drug(f.e. aspirin (acetylosalicic acid(sory for misspel)) right after you feel the symptoms of inflamation. I mean, there are no reasons to suspect misguided inflammation, or prelonged inflamation but it is still the most common way to treat a lot of diseases. Wouldn't it br better to let the organism usr the inflamation to cure the diseases, and use the anti-inflamatory if the inflamation prelongs, or it is very acute?

Popping pills for minor discomfort is rarely a good idea. People do things that aren't good for them. You might as well ask why people smoke or drink, since those things are worse for them than popping the odd aspirin. Also, in most of the world, aspirin is no longer the NSAID of choice, since it has potentially severe side-effects, including gastrointestinal bleeding. The name has sort of become genericised in the US though, to the extent that people call any NSAID aspirin, when in fact, it's far more likely to be paracetamol or ibuprofen.


text:
2 blue eyed children
My husband had light brown eyes. His father had hazel and his mother, light brown as well. His younger brother however, has blue eyes. Both my children have blue eyes. Is this possible? I thought once the recessive gene was used in my first child, his dominant brown gene would take over?

What is your eye color?

I think this - "I thought once the recessive gene was used in my first child, his dominant brown gene would take over?" is where you are going wrong.

There is some chance for that. Look at this chart with some probabilities:

Its from this webpage, which also gives some background information.
There is also an online calculation tool available, which takes the grandparents into account as well. It can be found here.

I think this color chart is dependent upon the heritage of the individual. (the population in genetic speak). there are no blue eyed people in some populations.  The mutation is only 10,000 years old or so. http://scienceblog.com/15361/all-blue-eyed-humans-have-common-ancestor/

I know. Its a mutation in the OCA2 gene, which is thought to come up from the black sea. See the answer in this topic for the reference.


text:
Does H. pylori have paracellular activity?
I know that it has transcellular activity i.e. it can pass through neighbouring cells.
However, I am not completely sure if it does not have paracellular activity, see this:

H. pylori did not induce any modification of the paracellular pathway
  (R = 148 ± 10 versus 174 ± 16 Ω · cm2; JNa = 4.16 ± 0.44 versus 3.51 ±
  0.41 μEq/h · cm2; JMan = 0.081 ± 0.01 versus 0.058 ± 0.009 μmol/h · cm2), nor did it modify J3H-HRP (2,201 ± 255 versus 2,110 ± 210 ng/h ·
  cm2 for H. pylori-infected and control cells, respectively). However,
  in the presence of H. pylori, we observed a significant increase in
  JHRPi (520 ± 146 versus 171 ± 88 ng/h · cm2). This effect was not
  dependent of the cag status of the strain and was not reproduced by
  the sonicates or the culture supernatants.

Is there any evidence that Helicobacter pylori has paracellular activity?

I found no evidence for paracellular movement.
Some notes of mine

Mucinase $\to$ lophotrichous flagella $\to$ Transcellular (actin fibrils, tails) movement
ICF Vacuolating cytotoxin (VacA) $\to$ altered T-cell function $\to$
  damages epithelial cells, disrupts tight junctions and causes
  apoptosis.

I think the latter point makes transcellular momevment possible.
I see no way how paracellular movement would be possible.

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text:
Do Yersinia genus spp have anything movement factors?
It can spread in blood (Type III secretion system -> apoptosis).
I am interested if there is any others ways to move.
Do Yersinia spp have anything virulence factors to move?

Yersinia enterocolitica does express flagella at a temperature around 25°C, but not at 37°C. Kapatral et al.
Besides that it can rely on peristaltic movement in the intestines.
Yersinia pestis is as far as I know nonmotile in the host, but motile if isolated Medical Microbiology.
As there are some other Yersinia species, including Y. pseudotuberculosis of course, I think you cannot totally generalize the answer for Yersinia spp., but searching for the Yersinia of interest with "flagella", "motility", "movement" or similar keywords on Pubmed might help.


text:
Evolution, self organization and neuroscience
I have rudimentary knowledge of evolution, and biology in general, so bear with me if this question is a bit naive.
Let's say we have a particular trait, like highly sensitive peripheral vision. There are two ways to go about explaining how this came about:
One is to say that evolution selected for it over time, because animals with a poor sensitivity to peripheral activity could easily become prey. So there is an advantage to having sensitive peripheral vision, and such a trait is adaptive. The picture I have in my mind is of an assembly line where there are randomly wired visual cortices and the best ones get selected for automatically. This seems to be the view of the field of evolutionary algorithms, at least to my mind.
Now, if we focus on the random nature of the wiring, we might see that there is an explosion of possibilities in terms of how the visual system can be wired together. After all, evolution is blind, and it doesn't know beforehand which physical signals are of interest.
I'm trying to think of a more plausible mechanistic explanation. Suppose we assume that given certain reliable properties of the physical world, like permanence of objects for example, evolution has found the optimal way to wire the visual cortex so that peripheral vision (and a host of other good-to-have features) is a natural outcome, then it would give us more focus in what environmental features and algorithms to look for. This can also be thought of as the idea that evolution, over time, has come to assume that certain things in the physical world will be a given and exploits these. (For example, objects in the peripheral areas of vision will tend to have different perceived velocities, so perhaps normalizing velocities across the field of vision automatically results in a need for higher sensitivity in that region. It just so happens that this could also be useful in detecting predators).
My question then is, is my assumption in the above paragraph valid? (Of course, the question then gets shifted to "Why normalize velocities across the visual field in the first place?").

I'm not sure that I fully understand the question, so I'm not answering. However the idea that adaptive evolution proceeds from a set of randomly wired visual cortices seems to me to be a wrong assumption - rather at each stage it starts from an already partially optimised wiring, with smallish changes offering some selective advantage. I think this resembles your more plausible explanation, but as I say, I'm not sure that I fully understand.

What is your assumption, I don't fully understand. You assume that given some properties of the world, evolution selects for the most optimal solution (for a given living thing to increase its reproductive success). Is this your assumption?

@Remi.b: My assumption, stated more simply, is a sort of "maximum efficiency hypothesis". That is, that evolution assumes the environment to be a certain way, and codes this into the developmental program, and therefore implicit in the developmental program are certain invariant features of the world, which the program uses to develop the brain fully. I think my question is more about developmental biology than it is about evolution.

@Joebevo See my update. let me know if it starts to answer your question! Sorry if I use some technical words that you don't know but their meaning are easily understandable from wiki articles. You might already know the concept of fitness landscape from computer science and genetic algorithm.

I recommend looking at Hoffman's interface theory of perception to help you explore some of the assumptions you are implicitly making in your question. Also, just because a fitness peak exists, doesn't mean you'll get there (for any reasonable notion of getting there).

another idea is that there are general algorithms for cognition/processing sensory data and they are reused in different parts of the brain for different sensory modalities (sight, hearing, etc) and all that is needed of evolution is that it "discovers" these algorithm(s), & incorporates them into neuron function. in other words why would peripheral vision processing be much different than "central" vision? this general idea/theme is somewhat articulated by Hawkins. see also research on visual "feature/line detectors" (possibly the basic function in play here)

fyi here is a recent blog on a candidate for such an algorithm supported by circumstantial/direct biological/computational evidence... line/feature detecting algorithm/sparse coding in visual cortex

@ArtemKaznatcheev: Thanks, that was quite an interesting video. However, I do think he's throwing the baby out with the bathwater. He's taking the so-called "bugs" in perception as evidence that perception is completely flawed. But, he does raise some interesting philosophical points about the truthfulness of perception, which is relevant to my question. (In general, I think throwing out conventional wisdom in favor of a computer simulation of evolution is dangerous).

Since the question is already answered, I will just add few points: Evolution is based on 'mistakes' (you can also call them 'experiments' or 'random-events' etc.). Experiments which are deemed useful are passed on to future generations and eventually selected. One additional point: in the context of your question, you are considering eye and brain (visual cortex) as separate, which is not the case. The eye is the sensor which sends brain all the sensory input data and brain processes it. The image you perceive, the colors etc. are your psychological response to different frequencies and inten

I don't fully understand your question. I hope the following will help a bit.
I'd like to pinpoint what seems to me to be one important mistake in your text. Reading you we have the feeling that selection acts on wires so to select the best combination. If this is what you meant, then it is obviously wrong. The thing is that the change that get inherited are alleles. An allele is a variant of a gene. For example: gene codes for eyes color. A bi-allelic gene has an allele which codes for blue eyes while the other codes for brown eyes. Assuming that there is genetic variance underlying the wire connection variance in the population, then yes selection will select for the best genetic variant. This does not necessarily mean that this best genetic variant is the most optimal possible to imagine solution. The most optimal allele might have never been created just because of the randomness nature of mutations.
EDIT:
To answer:

Do natural selection always bring a particular phenotypic (loosly speaking, phenotype = morphological) trait to its optimal state?

No! It does not.

The main reason is that the genetic variant coding for this optimal trait does not necessarily exist because of the randomness of mutational process. Also, because of the metabolic pathway bringing some variance to a trait, it is often very hard to even imagine a  mutation that would allow creating a new, more optimal, phenotypic variant. So indeed developmental biology has to be taken in account to.
Because in absence of genetic drift (infinite population), selection can only bring genotypes uphill in the fitness landscape. If a very high peak exist somewhere in the fitness landscape but in order to reach such peak, 10 mutations are needed were all of them in any other genetic background are deleterious (correspond to valleys in fitness landscape), then the optimal high will probably never be reached (except if all 10 mutations appear suddenly in the same individual).
Because selection might be weaker than genetic drift. Also, different subpopulations might be in different environments were different traits correspond to optimal traits and because of migration (gene flow) between subpopulations, it is possible that no optimal phenotypic trait can be ever seen although all the genotypic variance exist.
The seemingly most optimal trait might actually not be really optimal. For example: Peripheral vision might be limited by the brain rather than by the eye because it would be an important energy cost for the brain to process more information, a cost that might outweight the benefit of having a better peripheral vision.

Your two arguments are actually the same
The import point you are missing about the algorithm approach of optimizing a genome is it optimizes for the environment it was cycled through in the past. the laws of physics have been constant through those generations so the system becomes optimized for the laws of physics. Well optimized for physics at the scale of life at least, we are not good at handling relativity for instance.
It is much the same reason the human brain favors a desire for sweetness that is counter productive in a modern industrial world, because in the environment of our ancestors getting problematically high concentrations of sugar is simply impossible, so there is no benefit to having evolved a limiting behavior. Evolution has only had a handful of generations to select for anything else. Advantage and disadvantage are always relative to the environment, so the environment of the ancestral line effects how evolution optimizes an organism. Consider being very good at conserving heat would be very beneficial in the artic and very detrimental in a hot jungle. Retaining water would be very helpful in a hypertonic environment and very deadly in a hypotonic environment. Things are adaptive or maladaptive depending on the environment so evolution favors or disfavors the passing on of genes differently depending on the environment. A brain the predicts motion well under earth gravity has a big advantage, on earth, but since earth life evolved on earth brains can be slowly optimized for predicting motion under earth gravity, note I say can be sometimes the brain is stead simply made plastic and learns to predict motion in earth gravity because it is only exposed to earth gravity. It can be very hard to tell which is occurring.
Now it is worth noting optimization is only local not ultimate, Remi covers this quite well in his discussion of fitness landscape so I will refer you to it, I will just say optimized is not the same thing as perfect, it is only the best given what is available and given a history of X.

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text:
Why can we breathe only oxygen?
Plants has the capacity to take in both CO2 and oxygen, that's during day CO2 and at night oxygen. Why don't humans have such a capability?

Remember that O2 is also taken by the plants during the day !

You should have a look biological processes which are:

cellular respiration
photosynthesis

Loosly speaking, these two processes are just the opposite but deal with the same chemical compunds.
$C_6H_{12}O_6 + 6O_2 \xrightleftharpoons\  6CO_2 + 6H_2O (+ Energy)$
or in words...
glucose + Oxygen $\xrightleftharpoons\ $ Carbon Dioxide + Water (+ Energy)
From left to right is the process of respiration and from right to left is the process of photosynthesis.

Respiration releases energy from glucose (sugar). Therefore, animals, plants, fungi respire in order to get their energy from their food. More information on the chemical pathways on  Krebs cycle (or citric acid cycle). The main organelle involved in celullar respiration is the mitochondrion. Note: (Loosely speaking) the glucose is the end product of the digestion
photosynthesis allows organisms to create its own tissues from water and $CO_2$. But this cost energy. This is why plants use capture the energy form the sun with their leaves. More information on the chemical pathways on Calvin Cycle. The main organelle involved in photosynthesis is the chloroplast.

Note: In our lungs comes air which is a mixture of different gases (just like coke (Coca-Cola) is a mixture of different compounds). The two main compounds of air are Nitrogen (~78%) and Oxygen (~21%). We use only Oxygen. More info on the air composition here

Humans breathe in air, which contains both oxygen and carbon dioxide. But we do not photosynthesise and so we do not take in CO2. The CO2 produced from respiration in our body builds up in the blood and through diffusion escapes into the lungs and out when we exhale.


text:
Book on yeast reproduction, evolution and on the use of yeast as model organism
I am looking for a good book in:

Yeast reproduction

mating types
Recombination
mating type determination mechanism

Yeast evolution
Yeast as model organism and its use in experimental biology

It should include common technics used to work with yeast. For example: We can add a plasmid in order to fool yeast and let them think they have another mating type than the actually have.

Note: I am mostly interested in Saccharomyces cerevisiae of course.

Here is my list, in no particular order.
Methods in Yeast Genetics
Some other yeast books at CSHL
Fred Sherman's primer, available free at various sites including here.
The Freiburg manual here
The timeless classics (link to volume 3 but see all volumes)
The YeastBook is an attempt to build a current encyclopaedia to replace the previous item on my list.
The Saccharomyces genome database, probably the best organism-specific resource ever.


text:
Regarding kidney transplant
One of the options is correct

Kidney can be taken from a dying person who has the
a) cessation of neurological function only
b) cessation of cardiac function only
c) cessation of respiratory function only
d) cessation of kidney function only

I think that answer should be b. as if heart stops working then the person is definitely dead. but m not sure if on the cessation of neurological/respiratory function, one still has chance of revivial. Moreover answer is given as A. I don't know how. Could it be that on cessation of cardiac/respiratory function, kidney gets damaged while not so in case of neurological dysfunction?

This link describes the different ways of diagnosing/confirming death prior to organ donation. It might help answering your question.

I amn't really sure - but the answer given is A may be because there are many cases where cardiac function has been restored after hours of its cessation

There are actually very few situations where organs can be harvested from donors. For all deceased donor transplants, the donor must be confirmed as being brain dead (both brain stem and higher cortical functions).  However, in order for the organs to remain viable they must not become ischaemic - which is obviously a huge problem when the patients heart has stopped beating.  For this reason most patients who die and go on to become organ donors were already on a ventilator in intensive care in order to keep their blood oxygenated and circulating to perfuse their organs.
Kidneys are listed by NHSBT as a special case where a ventillator may not be required in advance to maintain viability - offhand I'm not sure why this would be - however to answer your question:
A. Neurological death - correct answer - the patient must be deceased without chance of recovery (hence brainstem and cortical death)
B. Cardiac function - were this to stop unilaterally then the patient may still be recoverable whilst the organs may be damaged due to lack of perfusion and thus not be viable for transplant
C. Respiratory function - almost identical to the cessation of cardiac function (and indeed will lead onto it very quickly)
D. Renal function - there's no point transplanting useless kidneys!
Have a look through the NHS Blood and Transplant Organ Donation site linked above, it's a great and trustworthy resource.

There's dead, and there's mostly dead.  Let's not give up on the "dying" pt who may only be in renal failure. :)

@AtlLED I'm not following you?


text:
How are lophotrichous flagella of helicobacter pylori produced?
I am thinking the mechanism is something like first adhesins (mucinase).
Howevever, this does not seem to be enough.
How are the flagella of H. pylori produced?


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Agglutination test using antibodies

Agglutination test
Latex agglutination using bound antigens : by coating soluble (non - particulate ) antigens on to microscopic latex spheres, their reaction with a particular antibody can be visualized.
Haemagglutination : red blood cells can be used as agglutinating particles.

What are non-particulate antigens ? Why are they used instead of particulate ones ?
How are red blood cells used as agglutinating particles ?

If you have an antibody that is directed against, for example, a bacterial surface protein, then by mixing the bacterial cells with the antibody at a suitable stoichiometry you could observe clumping of the cells as the antibody molecules essentially cross link the cells together. This would be an example of using an agglutination (clumping) assay with a particulate antigen. You can also probably see why it is important that each cell (particle) carries many copies of the antigen for cross linking to take place effectively.
If, however, the antibody of interest is directed against a soluble protein, then mixtures of antigen and antibody are less likely to form cross-linked complexes, although it can happen and can be used for example in the Ouchterlony technique. If you want to use an agglutination test (i.e clumping of particles) with a soluble antigen then you can achieve this by attaching multiple copies of the antigen to macroscopic particles. This is what is being described in the definition of Latex agglutination in your question.
I imagine that there are ways of attaching a soluble antigen to red blood cells (e.g. by chemical cross-linking to the red blood cell surface) in which case you could observe clumping of the red blood cells when you added your antibody.

From your answer,I infer that particulate antigens are those that are attached to a particle.

A particulate antigen is any antigen that is associated with a macroscopic entity (cell, synthetic particle) when it is encountered by an element of the immune system.


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Why is the number of PCR cycles limited?
I've been told that the maximum number of cycles in PCR is between 20 and 30.
Is this true, and what are the reasons for this limitation?

I would draw the line beyond 35, but thats a bit cosmetic.
The reasons are manyfold:

due to the exponential fashion of the amplification (ideally)
reagents are used up at some point
reagents degrade, this is especially true for the dNTPs
the activity of the enzyme, despite being heat-stable is declining
over time
beyond 35 cycles the exponential curve is flattening out (reasons see
above)
if you run the PCR for too long, you will get more and more
side-products (mostly primer dimers, but mis-aligned primers can also
make problems), this is more a problem for real-time PCR than for ones run on a gel

If you need a higher sensitivity with more cycles, you can use the technique called "nested PCR". There you do a first round with a primer pair specific for the region of interest and then do a second round with primers which are located slightly to the inside of the amplified DNA. This is done to avoid the amplification of unwanted contaminations. Since you do some 50-70 rounds of PCR amplification in total, this method is extremely sensitive (also to contaminations). See the image from the Wikipedia article for details:

Thanks for the answer! Would it then be possible to terminate the PCR, isolate out all the DNA and then start a new batch in PCR with all the isolated DNA, as to renew the polymerase and have "new" primers?

Yes, thats possible and is done. But to avoid the amplification of a contamination, you use a set of primers which is located slightly to the inside of the product and which does not overlap with the primers of the first round. This technique is called nested PCR (the article also contains an image of the technique.

I have added it to the answer.

How many cycles of PCR before dNTPs run out?
Assume a 25 μl reaction.
Assume 200 μM dNTPs.
200 μM dNTPs = 200 pmol  μl -1
so in  25 μl  reaction, there are 5000 pmol of dNTPs
= 5000 x 10-12 x 6 x  1023 molecules
= 3 x 1015 molecules dNTP
Assume that we start with 1 molecule of a 1000 bp template, 50% GC
1 kb = 2000 nucleotides
So , how many of these molecules can we construct using 3 x 1015 molecules dNTP?
= 3 x 1015/2000
= 1.5 x 1012 DNA molecules
How many cycles of PCR to produce this many from a single template molecule?
2n = 1.5x 1012
nlog2 = log(1.5) + log(1012)
nlog2 =  12.18
n = 12.18/ log2 = 41 cycles
Of course this in an absolute upper limit. The estimate assumes that you start with one template molecule of 1 kb, and that dNTPs aren't being hydrolysed, or otherwise degraded.

You have to take into account, that we use four different dNTPs for PCR, so this calculation would only be valid if the bases are equally distributed.

Which is why I stated the assumption of 50% GC (and so 50% AT).

You are right, I missed that point. Sorry for that.

In my own research I've found that the amount of PCR product increased as I increased the cycles to 50 times. This was for a single pair of primers used to amplify genomic DNA extracted with a particular kit as doesn't apply to other reactions I've done. What some people seem to forget is that the PCR products don't necessarily double at each cycle at any point in the PCR because some primers don't function as well as others and reaction conditions may be suboptimal (e.g. presence of contaminants or concentration of Mg ions).
In short, provided you aren't getting reduced-quality products (e.g. smearing or non-specific priming products), then I think it's fine to increase the number of cycles well beyond 30 - it's something you have to test empirically with your particular primers, template, polymerase, buffer etc.


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Is the Andraka pancreatic cancer test real?
In the wikipedia article about Andraka's pancreatic cancer test there are some controversial statements.
On one hand there are many glorious words about the method, also some awards, also some astonishing comparisons with other methods.
On the other hand, there is no proof, and also some evidence against its efficacy.
Also, I can't find any signs of Andraka's test in reality. For example, there is no such product in drug stores.
What is the proof or evidence that Andraka's method is really working and can feasibly exist in the near future?

Its mentioned in the Wikipedia article: None of the articles was published peer-reviewed and the replication of the results showed they were not significantly different from healthy persons.

Then what does award mean?

That some people, who probably have not much knowledge of scientific processes were impressed enough to award a price.

I think "fake" here implies that Andraka willfully misled people to earn the prize.  I don't think that is likely and speculating on it would be outside the scope of the site.  If you want a better explanation of the ways in which the method may not work as well as originally proposed, then you should re-word your question to reflect that.

@KennyPeanuts I have reworded my question. I don't care Andraka's motivation, only the reality of the method matters.

Basically, Andraka's method measures mesothelin. There's two key questions being asked about it (as it stands right now), falling into two categories:
1) There is some doubt as to the significance of serum mesothelin levels as a biomarker in pancreatic and biliary cancers. Some studies (e.g. Sharon et al (1)) suggest that there is no significant difference in levels - others (e.g. Hassan et al (2)) suggest that mesothelin may be a valid biomarker in some conditions but not others.
2) There are questions around the test itself. Andraka's work is promising and quite impressive. There are, however, some claims about it are being made that there's just not the evidence to support. I suspect that any fault for this lies with the journalists reporting on the story or PR people for various companies or institutions. The device may stand up to clinical trials and testing and be as revolutionary as it's claimed to be, but it does need to be fully tested.


text:
Natural examples of XOR functions at the cellular level
We can often think of cells as a sort of circuit on macromolecules, and can show that they can accurately and robustly implement functions like $\text{MAJ}(x_1,...,x_n)$ (return $1$ if more than half of $x_1,...,x_n$ is $1$ and $0$ otherwise) under natural conditions (Cardell & Csikász-Nagy, 2012).
Not all functions are created equal. $\text{XOR}(x,y)$ is a boolean function that is $1$ if exactly one of $x$ or $y$ is $1$ and $0$ otherwise (i.e, if $x = y$). It is often generalized to a multi-input function parity (or odd) as $\text{ODD}(x_1,...,x_n)$ which returns $1$ if an odd number of $x_1,...,x_n$ is $1$. Notice that this is superficially similar to $\text{MAJ}$ but is doing its counting in base 2, which one would expect to be easier for say finite state machines.
However, in some ways, this sensitivity makes the function much more difficult to the point that Valiant (2009) writes:

[Parity] appears to be biologically unnatural. That is exactly the prediction of our theory, which asserts that evolution cannot be based on the evolvability of [the class of functions parity belongs to]

Synthetic biologists used to agree to some extent, with Tamsir et al. (2011) writing that the closely related XNOR (i.e. $\text{XNOR}(x,y) = 1 - \text{XOR}(x,y)$) is empirically impossible to implement. However, they were proven wrong when Bonnet et al. (2012) implemented XNOR among many other amplifying gates by exploiting the structure and process of transcription. Unfortunately, this is an example from synthetic biology, and I really want to learn ones (if any) that occur naturally.
Are there any examples of XOR or related gates implemented (without human engineering) at the cellular level? Are there any naturally occurring molecular pathways that compute something similar to parity? (i.e. something similar to the majority result, but for a more 'sensitive' function) I am primarily interested in empirical demonstrations, but I would be satisfied with theoretical examples that are appealing to biologists (i.e. not something purely comp. sci. like P-systems or something).

References
Bonnet J, Yin P, Ortiz ME, Subsoontorn P, & Endy D (2013). Amplifying Genetic Logic Gates. Science, 340(6132): 599-603. PMID: 23539178
Cardelli L, & Csikász-Nagy A (2012). The cell cycle switch computes approximate majority. Scientific Reports, 2: 656. PMID: 22977731
Tamsir, A., Tabor, J. J., & Voigt, C. A. (2011). Robust multicellular computing using genetically encoded NOR gates and chemical wires. Nature, 469(7329), 212-215.
Valiant, L.G. (2009) Evolvability. Journal of the ACM, 56(1): 3-22.

Benenson (2012) also offers a synthetic example. http://www.nature.com/nrg/journal/v13/n7/fig_tab/nrg3197_F6.html I'll leave the answering of your primary question about natural examples to the actual experts. I am but the greenest of dilettantes in this field.

@JonathanVanMatre thank you, that's an interesting paper. Although it seems like they are implementing XOR through several cells working together with NOR gates as primitives. I will take a look at it, maybe they discuss some natural counterparts. All the synthetic examples suggest that it isn't unreasonable to expect a natural one, I think.

What's interesting to me is that it's not hard to come up with XOR gate implementations that sound biologically plausible: gene X is activated by two separate activators A and B. When present at the same time A and B bind each other strongly in such a way that their DNA binding domains are occluded, decreasing the transcription of X. It would be a leaky XOR gate, but it seems pretty biologically plausible. But my cursory search didn't yield much on competing trans-activators that can heterodimerize...

Empirical examples can be thought of but whether natural examples exist or not depends on the usefulness of XOR gate in biological systems. Considering a typical application of XOR, I can't see where a cell would want a binary adder. If we find an application we would find the example too. Can anyone think of its application ?

@WYSIWYG what are the empirical examples you have in mind? Are they still from synthetic biology? Right now I am following some leads by looking at mechanisms of co-inhibition in T cells, but my lack of knowledge (and other commitments) are making my progress slow. I am not sure if we have to look for binary adders, necessarily. That is just a particular way of thinking about it. However, if we want to go the adders route then base doesn't matter, any prime will work, so an application could be time/season/period tracking.

@WYSIWYG Keep in mind, that a clock/counter is not that great an analogy, since we want the circuit to combine several different input wires/signals and not just track one.

reminds me of mcculloch-pitts model from the 1940s. one could probably find (somewhat forceful) critiques of this model in the literature (mostly as too simple).

@ArtemKaznatcheev.. by empirical i meant engineer-able. However I see a situation where XOR might find a role but am not sure if the gate exists.
Morphogen/Chemokine gradient: if A = left to right gradient, B = right to left gradient...and output is defined as cell migration/development pattern then a XOR gate is implemented as A=1,B=1 will not produce a gradient for the cell to respond.

@ArtemKaznatcheev.. In fact I also have a hunch that lymphocyte activation is a likely place where XOR logic is used. I found another example: can't call it a perfect XOR Gate.. but the study says that combination of multiple antibiotics is actually not doing the job well compared to single drug treatment. Somewhat close... *PS: please let me know how adders can track period and wrt any prime numnber (it just increases the number of bits right. Pls fwd me texts on this if you can.Thanks)

Many sex-specific genes exist in a pretty much XOR state. Same for cell fate specification. Delta-notch and neurogenin could be an example, though it's not cell autonomous.

When I think about your question of natural examples of XOR, it pushes me to think about what type of natural environments (i.e., evolutionary pressures) would lead to the selection of an XOR equivalent.
When we implemented a synthetic XOR by "double flipping" one transcription terminator as a type of gene expression "check valve" it was the case that I did not know of any natural examples of flipping terminators, whereas I know of several examples of nature flipping promoters.
Why do we have plenty of natural examples of flipping promoters but (unknown to me at least) as yet no charismatic examples of naturally flipping terminators?
Perhaps we haven't been looking in the right environments to find them?
E.g., for a terminator to exist in nature, there needs to be something that must  be expressed upstream of the terminator, and also something that should not be expressed downstream of the terminator.  Since there are plenty of terminators there must plenty of examples of nature selecting for terminators.  My favorite example is the "Tø" terminator in phage T7 that is just downstream of the major capsid gene (highly expressed) but just upstream of a minor particle protein (weakly expressed).
But now reason thru what would need to be true in order to select for terminator flipping.  I'd need all the requirements of a static terminator (above), and then another such pairing of selective requirements under different environmental circumstances for additional gene products encoded in the opposite direction on the complementary strand of DNA. Phew!
I'm not saying that it is impossible to imagine such a complicated coupling of selection requirements emerging in nature, but it definitely seems a much more improbable aggregation of selective pressures to co-mingle all within a single genetic loci.
I'm still nevertheless very curious and would be grateful to learn of any natural examples.  -DE


text:
What is the current consensus, if any, about why long term potentiation is not clearly correlated with learning?
I was watching a video where a professor said that according to some experiments, Long Term Potentiation (LTP) is correlated with better learning, while in yet other experiments, LTP is correlated with poorer learning.
Are there any models that explain this phenomenon? What's the current theory on this?

Can you clarify what you mean by "LTP is correlated with learning"? Learning is a complex process and synaptic plasticity is certainly a necessary biological process.

I meant the strengthening of synapses. Some studies show that when synapses strengthen the learning is better, while others show that when they weaken, it is better. (There was no more detail given, but the professor just mentioned that it is an area that is controversial).

the idea that LTP is correlated with learning goes back decades. it would help if you could detail his statements if he had any further info. what is "poorer learning"? iirc LTP incidence is known to be higher in the hippocampus which is correlated with long term memory formation. (re famous patient HM in psychology). also lets remember that the "learning" that is measured in psychology experiments may not be the same as LTP or that maybe humans are still learning stuff that is not measured by the learning tests vs whats expected (ie there is the problem of "false negatives" here).

@Joebevo learning may require strengthening, weakening, or both of synapses. It will depend on many things including the learning task, context, which synapses, and so on.

I'm going to answer your general inexact question with a general inexact answer. The consensus is that LTP is correlated with learning.
To clarify in more detail, studies have shown that when an animal performs a learning task that involves the hippocampus(which is important for memory), and the animal effectively learns, less LTP is seen in-vitro in brain slices from these same animals. LTP is said to be occluded or blocked since potentiation of those synapses have already maximized through the in-vivo mechanisms. This can be interpreted as learning affecting LTP or at least both acting on the same basic mechanisms of the synapse(strengthening and weakening).
See for ex.
Learning induces long-term potentiation in the hippocampus.", Jonathan R Whitlock, Science (New York, N.Y.), 2006;313(5790):1093-7

I think this needs to be expanded upon to be a useful answer

I expanded on this; still not sufficient?


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Cloning of a sheep.. Dolly?
Dolly the sheep was a clone. When she was born she was eight years old on her 1st day of birth, her mother (from which she was cloned) was also eight years old. What is a molecular reason behind this - that Dolly was eight years old when she was born?

If you look at the replication of end of chromosomes, you will see that one of the strand gets shorter every time. To prevent the genes from being disrupted, the ends of the chromosomes have repetitive sequences called telomeres which shorten with every replication but will prevent the disruption of genes.
As you can deduce from the above information, there will be a certain limit to how many times a cell can divide.So, it has been thought that any organism ages because of shortening of telomeres.
Gametes have the enzyme telomerase which will lengthen the telomeres to full length so that the zygote formed will have unshortened telomeres. But, in the case of Dolly the nucleus was taken from a somatic cell and so the telomeres did not lengthen. The age of Dolly at the time of birth was thus the age of mother from which the nucleus was taken.
See : Telomere

Thank you so much for answering...It is really helpful.. :)


text:
Bioinformatics : Drawbacks of using ORA(Overlap Analysis)
What do you think are the potential drawbacks/weakness of using ORA to explain distinction between two phenotypes.
I identified a few which were the dependencies of DE and the statistical method used to filter the starting DE list.
Any other weakness?
Thanks

ORA = over-representation analysis; DE = differential expression.

It really depends on the specific methods you're using. If you're using a pathway-based methodology, I'd strongly recommend reading Khatri et al (2012) (1), which provides a good overview.
The main issue that's not touched on in that work is that pathway-based methods are highly dependent on the pathway definitions used, and the contents of the pathways are not always what you might expect in terms of their topology (a good example is KEGG's Neuroactive ligand-receptor interaction (2), which contains a huge number of events that look like 'single ligand binds to receptor', with no downstream events).

Like Liam said, this sort of analysis is dependent on the database you use, which may not be adequate for what you are trying to accomplish. And as far as I know, these databases are at the gene, not isoform, level. So if a change in two conditions is a result of a change in which isoform is expressed, rather than the amount of expression, you will not detect it.
Also it treats genes as equals that are independent of each other but thats usually not the case. For example if p53, at the top of a pathway, is expressed differently,  that has much stronger implications than if a gene at the bottom of the pathway was expressed differently. Additionally, it treats each pathway as independent of each other, but again that is usually not that case and there usually is some overlap between pathways.
You can read more here http://www.ploscompbiol.org/article/info%3Adoi%2F10.1371%2Fjournal.pcbi.1002375


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Translation of mRNA?
We inserted an insulin gene from human into bacteria. Will translation of the gene (protein formation) occur in bacteria? If translation occurs then why does it occurs, give a reason for this?

In the context of this question it is useful to distiguish between the primary product of translation - a polypeptide- and a functional protein. The insulin polypeptide has to be post-translationally modified by the removal of a signal sequence, the formation of correct disulphides, and by proteolytic processing to remove the C peptide before it can be called insulin.

Yes, bacteria will produce human (or any organism's) proteins if you introduce their genetic material but there are a few things to consider.
First, the introns must be removed from the human genetic sequence. Bacteria do not have the machinery to splice out introns after transcription. This is typically done by using a viral protein to reverse transcribe mRNA, which already have the introns removed, into DNA which you can use to transform bacteria.
Second, you need to insert a Shine-Dalgarno sequence. This allows the bacterial ribosomes to bind to the mRNA they will produce, and translate the protein you desire. This sequence is not found in eukaryotic genes, so you have to make sure it was included either in the vector or in the PCR primers you used to amplify the gene sequence.

Although the genetic code is almost conserved among organisms there are some issues to take in account, among which:

Every organism has its own codon usage, i.e. the differences in frequency of occurrence in synonymous codons in coding DNA (that reflect the composition of tRNA abundances). So, for example, the codons UUU and UUC both encode for phenylalanine but - I'm making this up - UUU is common in human but rare in coli while UUC the contrary. If the coding sequence of insuline is rich of UUU, you probably need to change the codon UUU to UUC in order to avoid slow translation.
Also the aminoacid abundance can be very variable between two organisms. One of the ways you can handle the differences is by "tuning" the expression of the aminoacids. Here a table of aminoacids abundance in coli.
The regulatory sequences in coli are not the same as in human. In fact, prokaryotic translation initiation needs a purine-rich sequence upstream the (usually) AUG initiation codon called Shine Dalgarno. This sequence is complementary to the 16S RNA in 30S ribosome subunit.
Often peptide chains are subjected to various post-translational modifications. The insulin peptide, for example, once synthetized is cleaved by specific peptidases and disulfide bonds are formed. Because human and coli post-translational modification mechanisms are not the same, this can be a difficult problem to handle. Instead of trying to exactly reproduce the protein with all its modifications, often people search for functionally (and not structurally) identical proteins.
Also the folding process can be different, both assisted (by chaperones) and spontaneous (withouth chaperones). The folding can be influenced by a number of factors like molecular crowding, pH, ...
A lot of eukaryotic genes have introns: sequences removed before translation. Once splicing sites are known, this problem is easily solved by using a sequence withouth introns.

All the organisms share a common ancestor and have evolved from it. So they share quite the same machinery for transcription and translation. Codons are also interpreted in the same way in almost all the organisms.
So, yes translation should happen ( Assuming that the gene has been cloned in the right way.)

It depends on the regulatory elements controlling this inserted sequence in the bacteria, isn't it?

It works, and is actually done to make all the "human" insulin which is used by diabetes patients today.
This is because the genetic code is almost completely conserved (with only very few exceptions in nature), so bacteria use the same codons to code for an amino acid as in humans. So with a suitable vector (a ring of DNA which carries extrachromosomal genetic information) which has the ability to replicate independent from the cell and which has the correct promoters (start points) for initiating transcription in bacterial cells, you can basically clone and express any human gene.

Is this question have any relation with degeneracy of genetic cods..???

Sorry, but I don't understand your comment? If you are asking, if the degenerated code is important here, my answer is no.

Ok.Then the Translation occurs in bacteria, because the genetic codes are  universal.

Yes, the code is universal (for exceptions see the Wikipedia, the regulatory elements are specific for bacteria (and need to be compatible).

Thanx for the help..


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Why do the humans become sleepy after meals?
I don't know about all the mankind, but I know enough people, who becomes sleepy after their meals. Also, I'm not sure, what kind of food do they consume, but I personally get sleepy almost from any food: sweet, fat, spicy, salty, liquid, tasty, not tasty, etc.
Is this phenomenon well known? And, if yes, how does it occur?

The functions of Parasympathetic nervous system - Rest and digest !

The name used frequently for this phenomenon is "Post-lunch dip".
"The post-lunch dip is a real phenomenon that can occur even when the individual has had no lunch and is unaware of the time of day. This dip has its roots in human biology, and may be linked to the size of the 12-hour harmonic in the circadian system. It is certainly exacerbated by a high-carbohydrate lunch, and may be more likely to occur in extreme morning-type individuals."
PubMed Source
Another PubMed source
"The 'post-lunch' dip is a bi-circadian phenomenon, largely unrelated to lunch, and worsened by a disturbed prior night's sleep. Despite anecdotal claims of adverse effects of larger lunches on afternoon driving ability, there is little actual driving data to support this belief."
For other reading and research...

What can be done to avoid Post-lunch dip? @Ilan

Coffee drink would improve the feeling I thing. In addition, meal with low glycemic index will lessen the phenomenon as well.

A relatively simple answer to explain your observation (yes, its not just you, it is something that actually occurs):
Orexin (or hypocretin) is a neuropeptide that controls arousal, wakefulness and appetite (Davis et al, 2011). As you might have understood, it acts as a critical link between what you eat and how much you sleep.
Generally, all kinds of foods contain glucose (ignoring some very rare examples, none of which I know honestly). When this glucose enters your bloodstream, it acts on not only insulin levels, but also orexin levels. Glucose has an inhibitory effect on orexin production, similar to leptin and opposite to ghrelin (Inutsuka et al, 2013).
Now, since increased orexin levels promote wakefulness, inhibition of orexin production causes sleepiness (Mochizuki et al, 2003). This helps explain how almost all kinds of meals cause a person to become sleepy.
References:

Davis JF, Choi DL, Benoit SC (2011). "24. Orexigenic Hypothalamic Peptides Behavior and Feeding - 24.5 Orexin". In Preedy VR, Watson RR, Martin CR. Handbook of Behavior, Food and Nutrition. Springer. pp. 361–2. ISBN 9780387922713.
Inutsuka A, Yamanaka A. The physiological role of orexin/hypocretin neurons in the regulation of sleep/wakefulness and neuroendocrine functions. Frontiers in Endocrinology. 2013;4:18. doi:10.3389/fendo.2013.00018.
Takatoshi Mochizuki, Thomas E. Scammell, Orexin/Hypocretin: Wired for Wakefulness, In Current Biology, Volume 13, Issue 14, 2003, Pages R563-R564, ISSN 0960-9822

What should we do to avoid it? @another 'Homo sapien'

@Pie not consuming heavy diets or meals rich in carbs might prove effective (I'm no expert in foods), or maybe a bit of caffeine (I wouldn't recommend it though, due to its addictive effects)

As far as I know it happens due the histamine release after meals. It leads to vasorelaxation and blood flow to the gastrointestinal tract. So, brain does not receive enough blood and you feel sleepy.

Please add some references for your answer.

Digestion burns a lot of energy as many muscles enter the mechanism as well as many chemical reactions.
Therefore, you feel tired as your body has less energy for other functions than it usually do.
I remember a lecture at the university about sciences' history in which they explained an experiment, a long time ago, led on death sentenced men. After a very good meal, one guy was going out for a ride while the other was going to sleep. After 6 hours, they killed both of them : the one who slept had fully digested the meal whereas, for the other, digestion was not complete. They guessed it did not go as well as part of the energy needed for digestion was used for skeletal muscles and that sleeping afer eating helped for a better digestion.

That is a very biased experiment, to say the least. Why, for example, do people then go for a "digestion walk" and actually feel it helps them to digest?

@skymninge: pure speculation, they mistake the effects on their metabolism of exercise, for the effects on their metabolism of having completed digestion. Or repeated contraction of the abdominal muscles while walking helps shift wind that they associate with poor digestion. Doesn't really matter why, the conclusions of a study of two people can be explained or waved away with any old crazy theory ;-)

Source : Here
Sugary foods lead to the production of insulin. Insulin stimulates cells to take up glucose from blood. It also mobilizes tryptophan to brain. Once in brain, tryptophan triggers formation of serotonin, a hormone involved in controlling mood and slumber.

the best sources are PubMed publications. Any other source are unreliable, especially site not related to any scientistic research and activity.

@llan Ohk..Will search and add some reliable references.


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Do "transcription factor binding site" and "operator" mean the same thing?
Can the terms "transcription factor binding site" and "operator" be used interchangeably in all contexts when referring to a DNA sequence, e.g. regardless of a cell type, whether or not the binding sequence is synthetic or natural etc.? If not, what are the differences?

As you no doubt know, the term operator was coined by Jacob and Monod as part of the formalism they developed to explain the properties of certain mutants in the lac operon in E. coli. In physical terms it is indeed the site of binding of a transcription factor, the lac repressor.
My understanding is that technically it is best to restrict the use of this terminology to bacterial systems in which the idea of an operon is useful. I don't think it would be wise to extend it to cover any site in eukaryotic DNA where a transcription factor is shown to bind.

To add about it: I am working with transcription factors for a few years now, I have never heard of anybody using it in eukaroytes. The sites are simply called transcription factor binding site.


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What would happen if you combined the world's deadliest diseases & viruses?
What would happen if you took the deadliest diseases/viruses in the world and combined them in a single medium (a solution of water or a test subject)? Would the strongest virus defeat the rest or would a  new deadly virus be created?

Viruses in the same cell can recombine (although they don't always do). This can happen by crossing-over of the genomic strands or reassortment of genomic segments. Therefore, you can get a new virus out of the fist two. The new one is not necessarily deadlier than the first two. If there's no recombination, the test subject will enjoy both diseases until the deadliest one kills him.

You're right : evil scientists are the best. You can start here : Viral Genetics
Have fun.

If you were to make a solution containing different viruses you would have a solution containing different viruses. Nothing would happen, they wouldn't "fight", they would just sit there. Drinking it would probably be a bad idea, but nothing would actually happen.
If you were to infect someone with different viruses then you would have given someone multiple diseases. Once again, they wouldn't fight, they would just go about their business infecting the host cells.
Now, it is possible that some sort of recombination would occur. Viruses work by inserting their genetic material (DNA or RNA depending on the virus) into the host DNA. Then, the cell's own machinery would start making copies of the virus. If a cell were infected by multiple viruses, it is possible that both would insert their genetic material into the host genome. If that were to happen, the likeliest outcome would be that the cell would simply start making copies of each of the viruses separately. It is extremely unlikely that any recombination would occur, but it is possible. In that case, assuming the viruses were similar enough, you might end up with a new virus, but there's no way of knowing if that would be more pathogenic, less pathogenic, as pathogenic as the original. Most likely it wouldn't even be viable and the combined genetic material of the two viruses would simply be inactive.
In short, viruses don't work the way you seem to think they do. They don't fight each other. They don't have anything resembling a mind. They aren't even alive by some definitions. It is not wrong to think of viruses as chemicals instead of as living things. They are incredibly simple and act following simple chemical rules. So yes, the likeliest outcome would be nothing interesting.

Kindly forgive my pedantry, but not all viruses integrate into the host genome.

@canadianer They don't? That's news to me, could you elaborate? Do some go into organelle genomes or something?

Most RNA viruses (except retroviruses) replicate in the cytoplasm. They can be translated directly by ribosomes and encode their own RNA-dependent RNA-polymerase.

@canadianer ah, of course. Yes, fair enough.

See viruses are made to destroy the host, not other viruses, so putting different types together really wouldn't have much an effect would it? as Manuella answer says, the host cell will simply suffer (or enjoy as he says) with both at once. Viruses don't mate so I sincerely doubt there would be a "combination". What some [evil] scientists are doing is that they take bacterium or viruses and they genetically modify them, and though this is a seriously tedious process, a bio-weapon could be created. Google bio-weapons and how they are made by governments.
Also, this would probably cost you a TON of money to do such a dangerous and complex project, and I doubt that anyone sensible would sponsor you.

Poison + poison = poison. They aren't integers to give you a result of 2poison. Viruses are a gatewat between living and non living organisms so they would barely mate and produce a more deadly virus. You might have been a fan of Umbrella Corporation (the company working as bio-weaponary making company which tests virus and takes over world) but in real life they don't work as we think they should.
Some virus would reverse the effect of the bad virus too. Thus as other answers has told you that being evil would cost you much more money than you think.
Bacterias are living viruses are both, so some would double up the disease whereas some might lessen down the effect of other virus.


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vestigial structure question
If a species has a vestigial anatomical
structure that it no longer uses, would
you expect this structure to disappear
over many generations through the action
of natural selection? Always, sometimes,
or never? Explain your answer.
I'm having a hard time with this question. I don't know what should I do

Its a homework question, right? Can you describe your basic ideas or what you understood?

I'm studying for an exam and the professor put this Q on a slide. I can't really remember what he said. I think the answer is Sometime. and there was something about recessive genes involved. Umm, I understand the natural selection concept and whatnot. But I can't gather enough infromation to answer it.

If a species has a vestigial anatomical structure that it no longer uses, it may or may not disapear through natural selection.
Evolution is based on four mechanisms :
- mutation
- natural selection
- sexual selection
- genetic drift
A no longer used structure can disapear if a mutation makes it to. Then, if it confers a selection advantage (natural or sexual), then individuals without that structure will survive and / or reproduce better than the other. The mutation will spread among the population and structure may finally disapear.
If it does not confer benefit the individual nor decrease it's chances of survival or reproduce compare to others, mutation may or may not be conserved and so for the structure.
Genetic drift can accelerate disapearance of a structure, especially if the population is really small, even if it does not confer any advantages to the individuals. The mutation can rapidly spread through the population just by chance.
For example, in human body, the appendix is no longer used but still remains. It's absence does increase chance of survivol nor is attractive to sexual partners, so it doesn't imply natural or sexual selection structure. Genetic drift may or may not randomly make it disapear one day, no one knows.

The appendix is not entirely vestigial.  It's a safe-house for gut flora following a bout of dysentery, and in places where dysentery is common, it  probably saves lives.


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question about directional selection
Cattle breeders have improved the quality of meat over the years by which process?
A) artificial selection
B) directional selection
C) stabilizing selection
D) A and B
E) A and C
I know the answer is D. It is obvious that is artificial selection but WHY is it also directional selection? how do we know? I know the differences between directional, stabilizing and disruptive selection but there is nothing in the question that explains the quality of meat in relation to directional selection. so HELP!

To help understand the problem, it might be good to restate the definition of directional selection that you know. What is directional selection?

There is a small issue in this question that is: everybody does not always use the exact same definition of stabilizing selection and disruptive selection. These two concepts are sometimes used to refer to phenotypic traits or to genes. For example stabilizing selection might either mean,

selection for the mean phenotypic trait of the population
or
selection for the wildtype allele, getting rid of all newcoming mutations.

Under simple genetic architecture the two concepts are equal.
Although for this question I don't think we even need to give a clear definitions between these two words, we'll use the phenotypic based definition (the first one above) in order to ease the intuitive understanding of the answer.
Let's say the trait breeders select for is mean quality and for ease again, we'll imagine this trait as being one unique trait that we can map on one axis from low quality to high quality. Here is a representation of that where the x-axis represent meat quality (left = low quality) and the y-axis represents the density of cows in the population which has the associated meat quality.

If breeders performed a stabilizing selection, it means that they would not improve the meat but only prevent the meat to change quality. In order to shift the quality of the meat to the right (to better quality), breeders need to perform directional selection.
I think the only problem that you had for solving this problem was the conceptual issue of considering meat quality just like any phenotypic trait. And if you want to shift a phenotypic trait on one side or another, you have to apply directive selection.

THIS WAS SO CLEAR :) I GET IT NOW!!
You are awsome and amazing.
Thank you very much.
Have a nice day, and thanks again for your time and efforts to construct this solid explanation :)


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What does 5' and 3' mean in DNA and RNA strands?
What are 5' and 3' in DNA and RNA strands? Please clarify with some images and please use simple English.

The 5' and 3' mean "five prime" and "three prime", which indicate the carbon numbers in the DNA's sugar backbone. The 5' carbon has a phosphate group attached to it and the 3' carbon a hydroxyl (-OH) group. This asymmetry gives a DNA strand a "direction". For example, DNA polymerase works in a 5' -> 3' direction, that is, it adds nucleotides to the 3' end of the molecule (the -OH group is not shown in diagram), thus advancing to that direction (downwards).

The five carbons in the ribose backbone are numbered starting from the O in clockwise direction* - therefore the carbon which has the base (A, T, C, G) attached is called 1'. The 2' carbon does nothing special, the 3' carbon has an OH group (downwards dash in the diagram), the 4' carbon connects the ribose ring to 1' via the O bond, and the 5' carbon is outside of the ring, attached to the 4' carbon on one side and phosphate on the other. PS: *) strictly, it's not after the ring direction but rather it comes from the linear configuration, i.e. when ribose is not in ring form.

@Armatus This comment is better than the answer.

The no 5 and 3 are the carbon no of the carbon skeleton ring of deoxyribose  as similar as any other organic compound. In any nucleic  acid, RNA or DNA 3' refers to the  3rd carbon of sugar ribose or deoxyribose which is linked to OH group and 5' linked to a triple phosphate group. So these  5' and 3' group provide a directional polarity to the DNA or RNA molecule. Now a good question would be y 3' and 5' not 3 and 5. It is simply to differentiate sugar carbons from that of the bases which are also having a carbon skeleton and thus nos for their carbon


text:
Effect of respiration on Arterial pressure
I am trying to explain the effect of respiration on arterial pressure.
I am looking preferably some visual way to show it.
How can you show the effect of respiration on arterial pressure?
I am thinking some kind of figure which has the respiration rate and arterial pressure.

I did a Google image search for respiration AND "arterial pressure" and found lots of graphs including one at http://69.36.35.38/accp/pccsu/update-evaluation-intravascular-fluid-status-critically-ill-patients?page=0,3 which may be what you are looking for

@AlanBoyd Your article is about venous pressure (outside of the topic).

Keep scrolling until you get to Figure 4.

@AlanBoyd I added two pictures about thing below which I think present the thing. What do you think is the most appropriate one?

Alan's pointing picture is this

I found this picture better

but not sure if it is exactly the level respiration to the arterial pressure.


text:
Late control mechanisms of blood pressure (arterial) level?
I first covered the fast and moderate control mechanisms which I think are

Fast control: Baroreceptive reflex (tonus), Chemoreceptive reflex
  (CO$_2$).
  Moderate control:  Bicarbonate
  (ECF), Phosphate buffer (ECF), Proteins (ICF).

then late control mechanisms about which I am unsure

Capillary fluid shift mechanisms
Stress-relaxation of the vasculature
RAA

What are the late control mechanisms of blood pressure?

Capillary fluid shift mechanisms
If the pressure of the blood is high, there is high hydrostatic pressure. Simply put, this means fluid exits the capillaries at the arterial side due to this pressure, and less fluid returns at the venous side. With less fluid in the circulation, blood pressure falls.
Stress-relaxation of the vasculature
Imagine the arteries are like springs. The more you pull them the stronger the recoil force. However if you stretch them too much for too long, they stay stretched.
Renin-Angiotensin-Aldosterone system
When the blood pressure is high, there is less release of renin from the kidneys. This means less angiotensin II production. Angiotensin acts to increase blood pressure by increasing vasoconstriction and increasing retention of fluid and salt (via aldosterone). With less RAA system activation, the blood pressure drops.

Can you say about the stress-relaxation of vasculature that high ICF leads to increased diameter and length of arteries?


text:
Converting unit/mg or unit/ml into ng/ml, or vice-versa
I am working with the enzyme Cu/Zn Superoxide (SOD1) and i am quantifying them in ng/ml.
However, most past studies has SOD1 quantified in unit/mg or unit/ml, and i need to compare the SOD1 level of my study with those past studies. Is there a way to standardize these units?
Thank you!

You need a value for the specific activity of the purified enzyme, in units/ mg (or similar). Also you need to check on the definition of a unit of activity for your enzyme since this is empirically defined.

There is no archaism in measuring enzyme amounts in units. Unit definition for SOD and a protocol can be found here (from Sigma): https://www.safcglobal.com/technical-documents/protocols/biology/enzymatic-assay-of-superoxide-dismutase.html :
"One unit will inhibit the rate of reduction of cytochrome c by 50% in a coupled system, using xanthine and xanthine oxidase ..."
I don't think, there is a good way to convert ng's to units, since one measures either mass or enzymatic activity, and the correlation between them is not always consistent. Units should be much more reliable in terms of comparisons with other studies, if you are not considering your enzyme as, e.g., a nutritious substance.

Quantification is a measure of your protein quantity and units/ml or mg is a unit of protein quantity. These are two different things. You need to set a rxn of SOD with its substrate and get activity which is proportional to your consumption of substrate. Then u will get the activity of your protein which will be in units/mg or ml

But: This is only valid for a certain batch, since the activity of protein preparations varies between batches. Thats why we have this units, which is not a very good thing. But better than nothing.

Right sir....:)


text:
Cranial Nerve Decussation
I want to get my head round this once and for all so if you guys could help me would really appreciate it! I understand lesions to the optic nerve. It is the other nerves that I find difficult.
So far this is my understanding, please correct me if I'm wrong. The cranial nerves do not decussate generally with exceptions being CN2, CN4, CN7 and CN12. That means if a lesion affects Cranial Nerve 1, 5, 8, 9 and 11 or the respective nucleus there is either sensation loss or atrophy of the muscle (dependent on what the nerve does) of the ipsilateral side. For example in a lesion affecting CN6, the eye cannot look out in the side of the lesion.
In the case of CN4, I'm not too sure what happens but I imagine if there is damage to the nucleus the opposite side is affected (meaning superior oblique in the contralateral side will not work). If the nerve is affected, it is typically the ipsilateral side. The same I imagine is true for CN12.
In CN7, the forehead is bilaterally innervated so if the upper motor neurone is affected eyebrows can be raised but the lower side of the face is affected ipsilaterally. In a lower motor neurone lesion, there is complete loss of one side of the face ipsilaterally.
Am I correct?

As you mentioned, the resulting impairment depends on the level of the injury: nuclear, fascicular, central / peripheral. So, you should differentiate lesions inside the brain tissue (which can be at nucleus, at any location of a tract, before crossing or after, and outside. From this, the first thing we should explain is the normal anatomy.  From normal anatomy we can understand the pathology.
One interesting fact you probably wanted to know is that Optic Nerve it not a real nerve in a classic definition, because it begins outside the brain as grouping axons of RGCs (retinal ganglion cells) and after leaving the eye becomes a “nerve”. From this, you can understand its damage better, because the damage to the visual fields depends on the level of insult.
IV nerve is the only cranial nerve that completely crossed (decussated). Thus, the lesion before the crossing/decussation (you were right mentioning the nucleus, but we extend the “region” until the decussation) will cause palsy and consequent atrophy contralaterally to the lesion. After the decussation the palsy and atrophy will be ipsilateral.
Considering cranial nerve XII we should speak about nuclear-infranuclear and supranuclear damage and with this notion supranuclear damage will cause contralateral transient tongue paresis. Nuclear-infranuclear damage will cause more permanent palsy, which is mostly bilateral because the nuclei are close to each other.
Considering VII pair, nuclear-intranuclear damage (“peripheral”) will cause ipsilateral hemifacial palsy including the brow and eyelid, while supranuclear (“central”) lesion will cause contralateral facial palsy with brow/eyelid sparing.

I just wanted to add, regarding CN VII.
CN VII receives stimulation via the facial nucleus from both the ipsilateral and the contralateral corticobulbar tracts. The upper muscles of facial expression (particularly around the eyes) receive stimulation from both the ipsilateral and the contralateral corticobulbar tracts. The lower portion of the face is innervated only from the contralateral corticobulbar tract. So in case of a stroke which wipes out an entire track (either left or right), there will be total loss of the lower muscles of facial expression, however there will be some sparing of the upper muscles because of the ipsilateral pathway redundancy.

Classically, only the CN.IV "decussates" (at the superior medullary velum) but some cranial nerve nuclei are on the contralateral side, so a lesion to the upper moto neurone or to cranial nerve nucleus of some cranial nerves will cause a contralateral deficits.
This is seen in cranial nerves VII & XII
Lesions at the lower motor neuron of cranial nerves will always cause an ipsilateral deficit.
As Daniel has pointed out, the upper motor neurons (corticobulbar tracts) that serve the facial nerve  (CN.VII) send fibres to synapse with both ipsilateral and contralateral lower motor neuron of the facial nerve, hence, the redundancy. This redundancy unfortunately does not involve the facial nerve that supplies the lower face (from the lower lip downwards).
If there is a lesion to the upper motor neurone that supplies the facial nerve on one side, there will be a contralateral paralysis of only the lower lip but not of the upper face because of the redundant supply to the upper face by the upper motor neurone on that side of the face.
A lesion to the lower motor neuron of the facial nerve as in Bell's Palsy will result in an ipsilateral paralysis of both the upper face and lower lip.

thanks for your contribution... though if you could add some external sources for validation it would be greatly appreciated. This is mostly for individuals to read further who do not have a good background in the question/answer.


text:
Does this equation have a typo? (Systems Biology, singular value decomposition)
I'm reading a systems biology paper, and I'm suspecting there's a typo in an equation, but I want to make sure.
The article is "Systemic metabolic reactions are obtained by singular value decomposition of genome-scale stoichiometric matrices." (Famili & Palsson 2003, http://www.ncbi.nlm.nih.gov/pubmed/12900206 / http://www.sciencedirect.com/science/article/pii/S0022519303001462). In equation 5, I wonder if the final subscript u_kr shouldn't be u_km, as the vector u_k^T is a column vector in the matrix U, which has dimensions m x m. Of course, some elements in u_k might be zero, in which case there would be less than m products in the sum, but I see no reason why there would be maximally r non-zero elements either (where r is the rank of the matrix S which has been decomposed). I worked out a simple example on paper where r < m and still got vectors with m non-zero elements. Can someone give me a second opinion on this?
Edit: Here are the main equations.
A model of a metabolic reaction network is described by a stoichiometric matrix S of dimensions m x n and rank r. For the metabolites in the model, changes in metabolite concentrations is equal to S multiplied with the vector of reaction rates, v:
${\frac{{dx}}{{dt}} = Sv}$
The singular value decomposition of S is given by
$S = U\sum {V^T}$
Multiplying on the left side with $U^T$ (The transpose of U) we get:
${U^T}\frac{{dx}}{{dt}} = \sum {V^T}v$
After matrix multiplication, we have for each row in $U^T$ and $V^T$, where $\sigma_k$ is the k'th singular value
$\frac{{d(u_k^Tx)}}{{dt}} = {\sigma _k}(v_k^Tv){\text{   k   =  1}},...,{\text{r}}$
And finally, equation 5:
${\text{u}}_k^T \cdot x{\text{  =  }}{{\text{u}}_{k1}}{x_1} + {u_{k2}}{x_2} + ... + {u_{kr}}{x_m}{\text{   (5)}}$

Can you provide the equation in your question, rather than asking people to go hunt it down?

I don't think there would be any point in presenting the equation by itself out of context. The equation is on page 3 of the PDF, right column - sorry for not mentioning that.

(a) this question has nothing to do with biology (although the paper obviously does, no biology is featured in the question) and (b) yes, it is a typo, as you can tell from it being $x_m$ and not $x_r$ and also as you can tell from equation 6 properly having $v_{k,n}$.

You are right this is a typo. Equation 5 is just a restatement of the definition of the dot product of two vectors:
$$\mathbf{u}_k^T\cdot \mathbf{x} = \sum_{i=1}^m u_{ki}^Tx_i$$
or written in another way
$$\mathbf{u}_k^T\cdot \mathbf{x} = u_{k1}x_1+u_{k2}x_2+\cdots+u_{km}x_m.$$
In the text Equation 5 is stating that one can find a linear combination of the metabolites described in $\mathbf{x}$ whose time evolution is described simply by a linear combination of the flux rates described in $\mathbf{v}$. The coefficients of the first linear combination are given by a row vector $\mathbf{u}_k^T$ in $U^T$. From this perspective, since there are $m$ possible metabolites ($\mathbf{x}$ is a $m\times 1$ vector), there have to be $m$ coefficients, so that the indices must range from 1 to $m$ in Equation 5. You are correct that $u_{ki}^T$ might be 0 for some $i$. But that doesn't change the fact that there should be $m$ (possibly vanishing) coefficients.


text:
How do you find pre-miRNAs from mature miRNA Blast output?
So in short, I did a blastn with standalone blast using all known miRNAs against the EST sequences in a genome (word length 7 and e-value 0.01). Now to confirm one of the next steps would be obtain the pre-miRNA sequence of each hit and do an mFOLD for structural confirmation. Each paper I read always mentions to get a sliding window (of 70 - 100 nt length) but how do you get this sliding window from the BLASTN output.. Do you have to code something yourself or is there an easier way to do it. Thanks.

miRNAs in EST- I am a little skeptical on this..

sorry for the late answer.. I am not sure if you still need it now.

DISCLAIMER: I have never worked with miRNAs.
However, from what little I know, the mature miRNA is produced by modifying the pre-miRNA. This means that you should be able to infer the pre-miRNA by mapping your miRNA to the genome using a tool like BLAT. Since you already have an EST which represents a transcribed sequence, you can map your EST back to the genome. You can simplify this by using a tool like exonerate that also models splicing.

A BLASTN would work for mapping the mature miRNA but you should correct some parameters.
E-value is fine but increase the word length. Keep it close to 17 because you are interested in perfect matches. With miRNA small mismatches can mean a different miRNA altogether. Also run for only plus/plus alignments. (You can see this post on biostar for details on how to do it)
What you can also do is to use bowtie (sometimes BLAST is very slow and though BLAST allows multiple threads there is no option for utilize multiple cores i.e true parallelization). Again use the option --norc to avoid plus-minus alignments.
Use bowtie-build on your EST sequences to make an index.
However, there is one more problem; it is unlikely that you'll get pre-miRNA sequences in your EST collection. This is because the stable form is mature miRNA and pre is generally too shortlived to be sequenced properly without appropriate selective measures.
Capturing small RNAs by sequencing is also not that straightforward. It needs size fractionation of RNA and subsequent library preparation from the small RNA fraction. So I guess you will not discover miRNAs from ESTs.
If you have small RNA sequences then you can align it to the genome and then select the $\pm 70$ nt to find the hairpin.
In fact there is an algorithm called mirdeep which does that; you can use it and avoid writing your own codes. If you are good at perl then you can modify it to suit your specific needs if any.

never used bowtie.. I am trying to work on blastn with the number of mismatches set to 2 at the moment. What would you suggest is a good clustering program to remove duplicate sequences

fastx_collapser will collapse redundant sequences. BLAST can get very slow for these things. It is better to use a short read aligner like bowtie.

thx.. will have to try bowtie maybe next time. Too far with the current project to retrack.


text:
Sequence analysis software suites
I have used DNAStar laser gene and VectorNTI in the past for cloning, primer design, sequence alignments, etc but no longer have access to these. Are there any comparable replacement suites or what stand alone packages would you now recommend?

TO do what? Please explain your actual requirements.

And for which operating system?

I should have specified OS. Either OSX or Linux. As for what I want to do with it, I need to be able to at least organize sequences, design primers from large sequences, local alignments, make plasmid maps, search ORFs, develop cloning strategies, etc. I also want to read chromatograms

All of this you can do with benchling (see answer below). And as it is web based it is not operating system dependent. As far as I know Geneious and GeneDesigner don't work in Linux.

CLC workbench is a decent program as well (see response below).

I would strongly recommend benchling https://benchling.com
This is an awesome web based tool for cloning, primer design, multiple sequence alignments and everything else you are used to doing with the other tools. It is very user friendly, and most importantly you can share designs with your collaborators. Also, the graphics are very beautiful. I recently started using it and I am really impressed with the technical support they offer: you can chat with them and they would reply within minutes. Also, if there is a feature you would like to have implemented they would definitely consider it. Benchling is free, open source and you might be even able to code up some features yourself.

My pleasure! Are you a benchling user?

Starting from today.

I personally use more frequently Geneious for most of the basic every-day manipulations (my university bought a license), but I would recommend Ugene: it's free, open-source, cross-platform and supports batching and scenarios.

I would recommend Galaxy. https://usegalaxy.org/
The system does include primer design, sequence alignments and covers many common bioinformatics tasks.

For primer design you can use the Gene Designer: https://www.dna20.com/resources/genedesigner
For sequence alignment you can use BLAST (from the NCBI, also downloadable as a standalone tool)
Of course there are better options that might be more specific for your situation.

If it's not loo late to contribute, CLC workbench is nice too (http://www.clcbio.com/products/clc-main-workbench/). I only use the free version (http://www.clcbio.com/products/clc-sequence-viewer/) but its very user friendly in terms of the features I have worked with.

Geneious is probably the strongest standalone package, but real bionformaticians rarely deal with GUI driven programs.

Why do you say that "real" bioinformaticians rarely use GUIs? Is it just some sort of macho "I'm too cool for that user-friendly stuff" attitude? REAL anythings get the job done with the best tools that are available for their particular problem domain. Sometimes it's text-based utilities, sometimes web-based, sometimes GUI-based. It's not about how much pain you can inflict on yourself, but whether you can get the job done correctly, on time, and on-budget.

@MattDMo It's not just elitism. For sequence manipulation, GUIs are much less efficient. They only make sense for people who have so little competence with programming that the added cost of learning this makes suffering a GUI desirable.

@Superbest please back up your statement with data that GUIs are "much less efficient." With today's modern multi-core computers and huge amounts of available RAM, the cost of running a graphical interface is not terribly high, considering that most users will be using one anyway. I am perfectly comfortable using Linux through the command line, and I am rather proficient in Python, but if pointing and clicking makes a one-off job easier than writing a new program for it, I'll do it. You say it's not elitism, but the rest of your comment is as elitist as they come.

They're not inefficient of resource usage, they are inefficient in terms of user time and effort. Many GUI tools are also critically underpowered. The Geneious next-gen alignment functions, for instance, do not output a SAM file (to my knowledge) that you can manipulate with other tools if Geneious functionality is not enough for you. Adding to that is the issue of citing proprietary algorithms in your publications.

@MattDMo Also, I'd like you to explain how my comment is "as elitist as they come".