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What determines a successful protein expression in E. coli?
Some proteins express well in a heterologous host; others- don't. A few requirements are known to determine the protein expression, like a strong promoter (like T7) for transcription and a strong ribosome binding site for translation. I am working with a protein, which consists of 2 subunits - alpha and beta. Both of them are on a plasmid with T7 promoter in front of the beta subunit (i.e. the construct is T7 promoter, CDS for beta subunit, CDS for the alpha subunit). The beta subunit expresses well, but the alpha doesn't. Do you thing this has something to do with the local environment (promoters, RBSs, etc) and how much does it depend on that? How can I increase the protein expression?

Some aspects are not entirely clear to me. The two subunits are expresses on seperate plasmids, or? And everything in the plasmids is the same except for the actual protein sequence?

The two subunits are on the same plasmid. The plasmid sequence starts with T7 promoter, RBS1, subunit beta, RBS2, subunit alpha. The RBS sequences are a little bit different, so I was thinking of making them the same, but this shouldn't have a great effect on the protein expression. (they are very close to the consensus sequence).

I found a very nice paper: Designing Genes for Successful Protein Expression, which covers most factors that determine protein expression. I post parts of it, because I am sure it will be useful to some of you.

Translation can be controlled at the level of initiation and elongation. Initiation of translation is primarily dependent on the sequence of the ribosome binding site (RBS) and early mRNA secondary structure. Other determinants of protein expression are less well understood but equally potent.
1. Initiation of translation
A key component affecting initiation of translation in prokaryotes is the RBS that occurs between 5 and 15 bases upstream of the open reading frame (ORF) AUG start codon. Binding of the ribosome to the Shine–Dalgarno (SD) sequence within the RBS localizes the ribosome to the initiation codon... Affinity of the RBS for the ribosome is a critical factor controlling the efficiency with which new polypeptide chains are initiated. This interaction is in competition with possible base-pairing interactions involving the RBS region that may form within the mRNA itself. Thus, SD sequences with weaker base pairing to the ribosome are more susceptible to interference from mRNA structure. However, some experiments suggest that SD sequences with too strong affinity can be deleterious, particularly at lowering temperatures, by stalling initial elongation. Also critical is the distance between the RBS and the start codon with 5-7 bases from the consensus SD AGGAGG being optimal.
Numerous lines of evidence suggest that the initial 15–25 codons of the ORF deserve special consideration in gene optimization. Studies have shown that the impact of rare codons on translation rate is particularly strong in these first codons, for expression in both Escherichia coli and Saccharomyces cerevisiae. In E. coli, peptidyl-tRNA drop-off during translation of the initial codons appears to be accentuated by the presence of rare NGG codons. These effects appear to be independent of local mRNA secondary structure. It is also true that expression may be recovered by 5' sequence replacement even for sequences that do not show especially strong mRNA structure or contain rare codons or other obvious deleterious elements in this region.
2. Codon bias
The second way in which host codon frequencies can be used is to match the host codon frequencies in the designed gene. This can be done simply by choosing each codon with a probability that matches the host codon frequency...Using sets of genes broadly varied in gene design features, Welch et al. found that variation in synonymous codon usage frequencies had a profound effect on the amount of protein produced in E. coli, independent of local 5’ sequence effects. Variation of at least two orders of magnitude in expression was seen due to substitution beyond the initial 15 codons of the ORF. This variation was strongly correlated with the global codon usage frequencies of the genes, although the codon frequencies found in the highest expressed variants did not correspond to those found in the genome or in highly expressed endogenous genes of E. coli. Multivariate analysis showed that the frequencies of specific codons for about six amino acids could predict the observed differences in expression. It is not clear what the biochemical basis is for this correlation.
3. mRNA structure and translational elongation
While much evidence suggests that mRNA structure can interfere with translational initiation in both prokaryotes and eukaryotes, the effects of structure on elongation are less well understood. This in part may be due to intrinsic helicase activity of ribosomes, which allows translation through even very strong hairpins and may preclude many structures from limiting the translation rate in either prokaryotes or eukaryotes. Perhaps more importantly, mRNA structure is difficult to predict, particularly for actively translated messages which are in continuous flux between various folded and unfolded states.
4. Protein-specific factors providing additional complexity
The protein may be particularly unstable in the host, especially if it is poorly folded due to inherent instability, lack of sufficient prosthetic factors, or improper post-translational modification... Expression of secreted and membrane proteins may be limited by mechanisms for directing these proteins to the membrane. It is even possible that the protein amino acid sequence may limit translational efficiency. For example, proline is thought to be slowly translated in E. coli, regardless of which codon is used.
Expression of the protein may be toxic to the cell leading to instability of the expression vector or host suppression of protein synthesis...A common strategy to reduce toxicity is to lower expression to tolerable levels. Promoters varied in strength can be valuable tools for finding an optimal expression rate for maximal yield...One potential way to avoid toxicity of some proteins is to direct expression to the periplasm or media. This may be accomplished by N-terminal fusion of a secretion signal sequence.

For more information, please read the whole paper. I also recommend reading Design parameters to control synthetic gene expression in Escherichia coli.

Thank you for the links! I've used DNA 2.0 for codon optimization & gene synthesis before and have had good results - I'm definitely interested in reading their publications.

@Amy: Yeah, part of the paper is about the features of DNA2.0, which I find very useful. I am a new user and still getting used to their software, but it looks pretty nice so far.

One important aspect when expressing a protein from a different organism in E. coli is that the codon usage of the original organism is likely different from the codon usage of the E. coli used for expression (Codon usage bias).
Though the genetic code is degenerate, not all codons are equal. They might encode for the same amino acid, but organisms tend to favor specific codons over others and the tRNAs for those codons tend to be present in different concentrations. When you now have a lot of codons in your sequence that are very rare in E. coli, the expression will suffer because the tRNA for those codons is not present in high enough concentrations to sustain the translation.
There are two ways of compensating for that, either you get your E. coli to produce more of the rare tRNAs, or you optimize your sequence to use different codons. For the first solution you can buy certain E. coli strains that contain plasmids encoding the tRNAs that are rare in E. coli, one of those strains is e.g. the Rosetta BL21 strain.
For optimizing the codon usage there are several companies offering to synthesize optimized sequences. There are also tools available online, but I have no direct experience with those.
The optimization is also not as easy as just using the most prevalent codon every time, it has been shown that optimizing the codons to match the translation speed in the original organism can enhance the expression yields. For some proteins, pause sites during the translation might be necessary to ensure proper folding. If the protein doesn't fold correctly it will be quickly degraded and your yield will suffer. This is described in the article "Heterologous Protein Expression Is Enhanced by Harmonizing the Codon Usage Frequencies of the Target Gene with those of the Expression Host" from Angov et al.
You'll find a nice overview about the whole topic in the article "You're one in a googol: optimizing genes for protein expression" from Welsh et al.

Thanks, Mad Scientist! Yes, I have thought about codon optimization and this is definitely on my to do list. Sorry I forgot to mention it. DNA 2.0 has a software which will convert your sequence into optimized one. I have also heard about Rosetta BL21, but I have never used it. We might also want to stick to our own strain, because it is the heterologous producer of of or drug of interest. I am a little concerned about codon optimization, precisely because of misfolding but I thing we should do it.

But this still doesn't explain why the other subunit expresses better, as well as other proteins from the same native organism are well expressed in E. coli. That's why I think it is more of a question about the control elements of transcription and translation, rather than rare codon usage.

@GerganaVandova You should compare the codon usage of your two subunit, it might be just by chance that one uses more rare codons than the other one.

I'm attending a talk by Claes Gustafsson from DNA2.0 - and i guess we knew this already - that codon usage is helpful, but doesn't always work.  they have a proprietary method which is more reliable...

Have you tried putting your two genes on two separate plasmids (with different origins of replication and antibiotic selection, of course) and co-expressing that way? If the first of the two subunits is expressing well and the second isn't, that's probably because the ribosomes are falling off the mRNA before fully transcribing the second gene. Are the genes eukaryotic in origin? I think it is difficult to "trick" E. coli ribosomes into treating heterologous sequences as an operon, but maybe someone with more knowledge of prokaryotic translation could help.
Actually, just adding a second T7 promoter in front of gene 2 would probably help quite a bit:

Genes are transcribed either from individual promoters or from a single promoter, leading to a long polycistronic mRNA. Polycistronic expression has been reported to lead to lower expression of the more downstream encoded protein, which can be exploited to influence the stoichiometry of a protein complex (9). Several-fold higher expression with individual promoters compared to polycistronic transcription has been reported (10). (Source)

I do know from personal experience that co-expression using two plasmids can work very well with the right genes!

Thanks for your answer. I like the idea of adding additional T7 promoter, although having repetitive sequences might lead to other future problems (I don't want to go into details). But I work with genes which are 3 times larger in size and one promoter for the 2 subunits works well. Perhaps it is very sequence specific.

Mad Scientist covered potential codon-bias issues (which can be ameliorated with Rosettas), but more generally, I've seen tremendous variability in expression rates between different vectors (pET-28 vs pET-24) without any apparent reason.  Our lab has had tremendous success with IPTG-inducible vectors (going from pBC-SK to pET-24 increased expression 50-fold).
Beyond expression, the protein can be lost when preparing the cell-free extract.  It may not be in the cell pellet if exported courtesy of a signal peptide, or may be discarded in the "debris" pellet when spinning down lysed cells if it's poorly soluble.  I've heard a theory that solubility issues may be exaggerated by vectors that saturate the export machinery, which can both impede synthesis and/or be so effective they just cause precipitation.  The pET System Manual suggests longer (overnight) expression times at lower (15-20 °C) can help solubility issues.  Whatever the reason, engineering away the signal peptide drastically increased expression for many of our proteins.


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What is causing my problem with very low yields when isolating a 42kb yeast plasmid?
I have to isolate a large plasmid from yeast and transform it in E. coli. After transformation, I often get no colonies. One reason for that is the yeast mini prep hasn't worked or the DNA concentration is too low. I tried two different isolation protocols:

Zymoprep I yeast plasmid prep kit
From the doc: "based on the old E.  coli alkaline lysis method with our Zymolyase added in the first  solution."
Phenol chloroform YAC isolation protocol
used by Dan Gibson et. al] for their huge constructs (>100kb)
They use an expensive Qiagen large-construct column at the end, in order to purify and visualize the yeast extract on a gel, but I skip that step because the Qiagen column cost $40 per yeast clone which doesn't scale if we do many of these.

How can I troubleshoot and optimize the above-mentioned protocols? Are there any other protocols that might be more suitable for a large plasmid and don't use too expensive materials?

A dependable protocol for yeast DNA extraction I used to use was broadly similar to the protocols you cite but included an ethanol precipitation before and after the P:C extractions. The only expensive material was time. The general outline was:

Alkaline lysis
Ethanol precipitation
RNase A treatment of resuspended DNA for 15 minutes
Phenol:chloroform extraction (x2)
Ethanol precipitation

You probably know that the ethanol precipitation lore is that the yield is better the longer and colder you do your ethanol precipitations. I only ever did reverse transcriptase reactions a few times, but the template DNA I prepared spent the night in ethanol at -80C to maximize yield. Another place to double-check is that your yeast cultures are sufficiently dense before starting.

What should be the OD of the culture? What is the volume of the culture?

I took 1.5mL of a "dense overnight culture" for this protocol. I don't remember being particularly careful about the OD for this (and the OD is relatively strain-dependent anyway). For us, "overnight" meant the culture was in a healthy stationary phase and that was easily gauged by eye. If you need to get a feel for the growth properties of your strain, start a culture in the morning and take periodic OD600 readings throughout the day. That should give you a clear picture of when you'll have the maximum number of healthy cells.

I've successfully used the Zymoprep kit, but only for smaller plasmids in 2-hybrid experiments. So their cell lysis enzyme/buffer system works well, but I'd guess that your 42kb plasmid is precipitating out with the genomic DNA.
The standard Qiagen midi columns are capable of capturing large constructs. This has worked wonders for me when I was purifying large BACmids from E. coli - and gets the cost down to $10/sample (it does require additional buffer, but that's a minor expense). It might be worth trying the Qiagen yeast DNA protocols for the midi kit and see if you can isolate your large plasmid?

And for what it's worth, the technical reps for Qiagen were very helpful on the phone. Unlike Invitrogen. I dread every call to Invitrogen technical service. >:[

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Why is thymine rather than uracil used in DNA?
What is the advantage gained by the substitution of thymine for uracil in DNA?  I have read previously that it is due to thymine being "better protected" and therefore more suited to the storage role of DNA, which seems fine in theory, but why does the addition of a simple methyl group make the base more well protected?

I have now posted the original question, "Why is uracil, rather than thymine, used in RNA?" separately, as promised.

One major problem with using uracil as a base is that cytosine can be deaminated, which converts it into uracil. This is not a rare reaction; it happens around 100 times per cell, per day. This is no major problem when using thymine, as the cell can easily recognize that the uracil doesn't belong there and can repair it by substituting it by a cytosine again.

There is an enzyme, uracil DNA glycosylase, that does exactly that; it excises uracil bases from double-stranded DNA. It can safely do that as uracil is not supposed to be present in the DNA and has to be the result of a base modification.
Now, if we would use uracil in DNA it would not be so easy to decide how to repair that error. It would prevent the usage of this important repair pathway.
The inability to repair such damage doesn't matter for RNA as the mRNA is comparatively short-lived and any potential errors don't lead to any lasting damage. It matters a lot for DNA as the errors are continued through every replication. Now, this explains why there is an advantage to using thymine in DNA, it doesn't explain why RNA uses uracil. I'd guess it just evolved that way and there was no significant drawback that could be selected against, but there might be a better reason (more difficult biosynthesis of thymine, maybe?).
You'll find a bit more information on that in "Molecular Biology of the Cell" from Bruce Alberts et al. in the chapter about DNA repair (from page 267 on in the 4th edition).

I always just assumed that ribose better attached to uracil and deoxyribose better attached to thymine. As far as you know, this is not necessarily true?

@fredsbend the 2'OH of the ribose and the methyl group of thymine are pretty far away from each other, there is no obvious way they would affect each other.

@user338907 either I don't understand how evolution works, or that paper doesn't (assuming you paraphrased it accurately). As far as I know evolution doesn't need to "see" any future possibility, it simple rolls the dice and whatever works get passed on.

I think it's justified and relevant, I'm just surprised to see that claim from professionals. I'm a layperson when it comes to biology so I'm sure they know better than I do. I would have though the U-T mutations could have occured, perhaps been useless and benign but persisting to some offspring, and then later the offspring developed the advantageous UNG enzyme and both mutations then propagated through the population. I suppose I don't see how some future knowledge of the advantageous result is required.

The existence of thymine in DNA instead of uracil is apparently due to evolution process which made DNA more stable.
Thymine has greater resistance to photochemical mutation, making the genetic message more stable. A rough explanation of why thymine is more protected then uracil, can be found in the article

Arthur M, L., Why does DNA contain thymine and RNA uracil? Journal of Theoretical Biology, 1969. 22(3): p. 537-540.

which gives three major reasons for it to happen:

"Excitation  energy  in  DNA  is  mobile,  and  is  eventually  transferred  to
thymine  residues,  which  are  the  sites  of  radiation  damage."
"Uracil but not thymine forms a stable photohydration product. The dimerization of thymine can be partially photoreversed by irradiation at relatively longer wavelengths, while this process is less effective for uracil dimers because of the competing photohydration reaction"
"Photochemical  mutation  is,  or  at  least  was  at  one  time,  a  serious
problem,  since  there  exists  a  series  of  enzymes  to  repair  radiation  damage.  Therefore  resistance  to  radiation  damage  was  an  important selective  advantage."

Thymine has a greater resistance to photochemical mutation, making the genetic message more stable. This offers a rough explanation of why thymine is more protected then uracil.
However, the real question is: Why does thymine replace uracil in DNA? The important thing to notice is that while uracil exists as both uridine (U) and deoxy-uridine (dU), thymine only exists as deoxy-thymidine (dT). So the question becomes: Why do cells go to the trouble of methylating uracil to thymine before it can be used in DNA? and the easy answer is: methylation protects the DNA.
Besides using dT instead of dU, most organisms also use various enzymes to modify DNA after it has been synthesized. Two such enzymes, dam and dcm methylate adenines and cytosines, respectively, along the entire DNA strand. This methylation makes the DNA unrecognizable to many nucleases (enzymes which break down DNA and RNA), so that it cannot be easily attacked by invaders, like viruses or certain bacteria. Obviously, methylating the nucleotides before they are incorporated ensures that the entire strand of DNA is protected.
Thymine also protects the DNA in another way. If you look at the components of nucleic acids, phosphates, sugars, and bases, you see that they are all very hydrophilic (water soluble). Obviously, adding a hydrophobic (water insoluble) methyl group to part of the DNA is going to change the characteristics of the molecule. The major effect is that the methyl group will be repelled by the rest of the DNA, moving it to a fixed position in the major groove of the helix. This solves an important problem with uracil - though it prefers adenine, uracil can base-pair with almost any other base, including itself, depending on how it situates itself in the helix. By tacking it down to a single conformation, the methyl group restricts uracil (thymine) to pairing only with adenine. This greatly improves the efficiency of DNA replication, by reducing the rate of mismatches, and thus mutations.

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How Do Large Ocean Viruses Form Their Own Organelles?
Several large viruses (Arslan 2011) form their own organelles within the amoebae they invade.
How do these organelles form?

Reference:
Arslan, D., Legendre, M., Seltzer, V., Abergel, C., Claverie, J-M. (2011) Distant Mimivirus relative with a larger genome highlights the fundamental features of Megaviridae. PNAS 108(42): 17486-17491 [DOI]

The organelle form in the same way they would if they were coded for by the host's genome, except in this case the virus uses its own genome to specify the type of organelle.
The article you link to indicates this.  The "systems" in the quote are in part the organelle that it needs

A study of the giant virus’s DNA shows it to have over a thousand genes, the biochemical instructions it uses to build the systems it requires to replicate once inside its host.


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How much time do INS1-E and MIN6 cells need after splitting?
I am currently doing an experiment on cells to test the internalization of a protein.
Normally, I seeded my cells the day before the incubation. This worked well for Hela, CHL or PANC1 cells. However, when I did the same with INS1-E and MIN6 (both beta-cells) after the incubation and the washing step the majority of the cells were gone. This was better in the control where I did not put in the compound, but still a lot of cells were detached.
Therefore, I wonder if I should seed the INS1-E and MIN6 cells earlier, more like 2-3 days before. Do these cell lines need more time after splitting to attach again?

I think that to adequately answer this question, we need to know the composition of your culture medium and any additive with which you supplement it. I'll assume 37 oC and normal oxygen levels. The size of the cultures makes a difference - spinner flasks are not the same as tissue culture plates.

The common formulation for INS-1 cells is to use RPMI media, and it's an adherent cell line. I don't know about MIN6.

Searching literature to see what the published protocols were is the best bet. I found a simple document for INS-1 cells here. There is also this paper describing the isolation of an Ins-1-derived cell line 832/13 which my lab (though not me personally) have experience with. Cells should only be sub-cultured when nearly or fully confluent. With Ins-1 cells that would be a sub-culture ratio of 1:4 to 1:8 and will take 2-3 days. Something to note is that Ins-1 cells are poorly adherent to tissue culture plastic. That's a normal part of their characterization. I remember lab members having to take special precautions with respect to media ingredients to prevent them from detaching, as well as really delicately adding media to the dishes otherwise the force from pipetting too quickly will dislodge many cells and explain why your cells are washing away.

I've worked with MIN6 cells in the past, and have found that they can take at least 12-24 hours to fully seed (if they are being cultured on polystyrine).  Depending on the specifics of your experiment, and whether doing so would complicate your controls, you might also want to try coating the surface you are attempting to seed on with laminin.  MIN6 cells seem to like growing on that over uncoated polystyrine and other ECM proteins.


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What is the effect of exendin on beta-cells?
Do you know if exendin, an analog of GLP-1 (glucagon like peptide-1), can be toxic for beta-cells? For example, what is the effect on INS1 or Min6 cells at a certain concentration or after 90 mins of incubation. And what is the impact on insulin secretion?

At first glance, it should not be toxic considering that it is a normal endocrine signalling pathway withing pancreatic beta cells (exendin just being a synthetic GLP-1 analog).

Old question, but new data: Protective effects of exendin-4 on ß cells incl. INS-1E lines (1)(2).

I recently tested exendin on INS1e cells in an Edu incorporation assay (similar to BrdU incorporation) to observe if this compound induces proliferation of the cells.  Compound incubation was for 24 hours.  I saw no incorporation of EdU with this compound over untreated levels.  In addition, during the assay I look at overall cell number with a DNA stain, but I did not see any effect on total cell number with exendin (ie, if it were toxic the total cell number should go down which I have observed with other compounds).


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Are there animal models for Clostridium difficile that better replicate human infection than hamsters?
So I'm looking for some information on the infectious dose necessary to colonize a human with Clostridium difficile. There's no human challenge studies, and since it's not a foodborne pathogen, little we can get from outbreak data.
Which leaves me looking at animal models, to at least get the shape of the probability of infection based on a given dose. But the papers on hamsters makes it appear that they're infected rather easily, and that this infection is particularly severe.
Is there another animal model that more closely mimics the infection dynamics of C. difficile in humans?

In any event... have you seen this?

Hamsters are considered the best animal model, most studied.
C. difficile has been found in calves, ostriches, chickens, elephants, dogs, horses, and pigs, but its role in infection and its pathogenesis in animals are largely poorly understood and possibly underestimated


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Is there an equivalent to "Fields Virology" for Bacteria?
I've gotten a staggering amount of use out of my copy of Fields Virology as a general reference for "getting me up to speed" on whatever pathogen I'm currently looking at. I don't know of a similar type of reference for bacteria, but I only became aware of Fields as I was moving out of the field, rather than into it, so it's possible I've simply never heard of a suitable reference.
Any suggestions?

I might as well give this as an answer: the closest analog to Fields for bacteriology that I'm aware of are the two Bergey's manuals: Bergey's Manual of Systematic Bacteriology, and Bergey's Manual of Determinative Bacteriology. They deal with taxonomy and identification of bacteria (and even cyanobacteria and archaeans), with some emphasis on the medically important classes.

The Manual of Clinical Microbiology from ASM is the bible we use in our Micro lab. It's often our first reference. There are a lot of other useful books in the ASM eStore.
ASM eStore


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Does the oxygen concentration equilibrate between red blood cells in the liver sinusoids?
In the sinusoids of the liver, venous blood from the hepatic portal system mixes with arterial blood from the hepatic arteries.
Do oxygen molecules move between oxygenated RBCs and non-oxygenated RBCs, so that the partial oxygen saturation of the blood in the sinusoids reflect partial saturation of the RBCs? Or do RBCs retain their initial oxygen saturation, so that the partial saturation of the blood in the sinusoids reflects a mixture of saturated and unsaturated RBCs?

I am not sure if these are the best tags, feel free to adjust them is they can be improved.

The oxygen saturation (in lungs) and desaturation (in target organs) takes place via diffusion along the concentration gradient (i.e. partial pressure for gases). Therefore as long as RBCs from two different sources and having different partial pressure of oxygen mix up, the oxygen level starts to equilibrate between these cells.
But diffusion as a passive mechanism is not very fast and can take some time. So the only possibility for the second variant (there is a mixture of two types of RBCs) is if the mixture exists for very short time in liver sinusoids, so that the blood leaves them without really mixing completely up.
This is not true, at least in some animal models the blood within the liver seems to reach equilibrium very quickly and its resulting partial pressure can be influenced by adjusting the blood flow from different sources (using vasoactive substances injected directly, as in the referenced paper).

I don't understand why you think that diffusion wouldn't be fast enough, since it is the same mechanism that ensures that blood becomes oxygenated in the pulmonary circulation, and deoxygenated in the peripheral tissues.

@AlanBoyd is right to be confused. This answer is unsourced and based on statements that are generalizations and, when applied to this question, false (diffusion of oxygen is is not fast enough to equilibrate within a blood vessel).

I'm not aware of any studies that investigate the equilibration of oxygen saturation between two individual cells in hepatic sinusoids, but I will answer based on what we know about these issues in the general context.
See West, Pulmonary Pathophysiology, Ch 2, oxygen loading in a healthy lung occurs to full saturation in about 0.25 seconds, one-third of the available contact time:

This involves diffusion through surfactant, the alveolar membrane of an alveolar cell, the cytoplasm of an alveolar cell, the basal membrane of an alveolar cell, the basal lamina, the basal membrane of an endothelial cell, the cytoplasm of an endothelial cell, the luminal membrane of an endothelial cell, plasma, and the membrane of an RBC. (see this illustration from Ross Histology)

In the liver, oxygen rich blood arrives from the hepatic artery, nutrient rich blood arrives from the portal vein. Blood from these different sources mix directly in the same vessel in the same hepatic sinusoid. There is no alveolar cell, basement membrane, or endothelial cell in the way. Oxygen just has to diffuse across one plasma membrane, in blood, and across another plasma membrane, all in a fluid moving together.

Diffusion of a small molecule like oxygen is quite fast, and RBCs are made for rapid diffusion, even of a large globular protein like hemoglobin.

Good answer overall; I think it might be worthwhile to quantify the speed/distance of oxygen diffusion, though. Oxygen does indeed diffuse quickly over short distances, but in tissue the oxygen concentration falls off quite rapidly with distance. RBCs are simply quite small, and I think this is at least if not more important than the rate of diffusion.

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Recommended applications for commercial antibodies
Commercial suppliers of primary antibodies for given protein targets typically list recommended applications, for which the antibody has presumably been shown to work. I am usually looking to use them for immunofluorescence, and my impression (which admittedly may be based on an unrepresentative sample) is that most antibodies are recommended only for use with Western Blotting.
Why is this? Is WB the most common application? The most general? Is it easier to test for than IF?
And, perhaps more to the point, how seriously should one take these recommendations? If an antibody is not recommended for IF, does that mean that it probably won't work? Or that it may well but no-one's bothered to check? Or that it almost certainly will?

Maybe WB is easier for them to test?
In general, however, my recommendation is to check if they provide refs to paper using those antibodies and look at the images on the paper.
Be careful putting your trust in the supplier's pictures, I have seen obviously photoshopped images on commercial websites.
Also always double-check the referenced papers, as sometimes they don't really use the same product that is sold...
Other than that, in my experience, the recommended application is just that: recommended. The antibody may as well work for other application.
That said, note that there is a rationale for an antibody working for WB and not IHC, for instance: perfusion with fixators such as PFA will change the 3D structure of the target protein and may mask (or reveal) certain epitops, so the antibody may be more or less efficient depending what form of the protein you're dealing with (native, fixed in PFA, fixed in glutaraldehyde, SDS-denaturated etc.)

Antibodies for WB and for IF have fundamentally different requirements for the epitope structure. Antibodies for WB recognize denatured structures extended by SDS treatment. Antibodies for IF often will need to recognize a native-like structure.
Depending on the supplier, some will have lots of antibodies for IF. I suggest looking around some more.


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Is telomere length a reliable measure of health/lifespan?
Several companies are commercializing tests for telomere length such as this one here. I understand the basic mechanism for why telomeres shorten during DNA replication, but how good is the evidence that telomere length is a reliable indicator of healthspan/lifespan?

I disagree with Ctina.  So many factors go into determining loss rate, activity of telomerase, gene conversion or unequal exchange at chromosome ends, etc to ever say that length is a function of age.  There is far more variation between individuals and between cells within an individual than aging ever shows.  Here is one study:
Das B, Saini D, Seshadri M. Telomere length in human adults and high level natural background radiation. PLoS One. 2009 Dec 23;4(12):e8440.
however I've seen similar studies published elsewhere.  Telomere length as diagnostic for age is very poor at best.  Here's an article that gives a formula, but acknowledges the R2 is 0.04, which is useless: http://preview.ncbi.nlm.nih.gov/pubmed/18378481

There's no math equation processing at the moment but you can use the html  tag if you wish for things such as R-squared =)

Please note that I did never say that it is reliable.
So I don't understand in what you disagree exactly. I also referred that are many factors that alter the size of the temolere, as you did.
Just because I did not stated "It is not reliable" I also did not say "It is reliable". That was my mistake to not make this point clear.
But it is true that it gives a rough idea of lifespan (I said rough, not accurate!)

I disagree that "It would be reliable if we don't take into account the environment, and if we are comparing two genetically identical persons." There is far too much stochastic change (and non-telomerase-based repair such as interchromosomal exchange at telomeres).  Two cells from the same individual are not concordant, so your statement cannot be right. To say that it is "not 100% reliable" suggests it's still pretty good (but not perfect). It's less-than-4%, so I disagree that it can be used for "a rough idea."

Ok, what you said is true. agreed. vote up

A number of studies have compared replicative capacity of cells with lifespan.  Since Replicative capacity of cells is linked to telomere-length, these studies may provide indirect evidence for the association between telomere length and lifespan.  One interesting study in particular compared animal life spans and in vitro replicative capacity of skin fibroblasts in groupings of small, middle, large, and very large breeds of dogs of specific ages (Li et al, 1996). It was found that the life spans were inversely correlated to the frame sizes of the breeds. It was shown that all the small breeds studied have a longer life span than that of the large breeds. The replicative capacity of fibroblasts from the large dogs (Great Dane and Irish Wolfhound) was significantly decreased compared with that of the small dogs. The reasoning behind these observations may again be due to varying degrees of cell turnover between the species. Large dogs consist of more cells than small dogs and as a result more cell turnover was initially required in their development compared to small dogs. This increase in cell turnover would subsequently lead to a decrease in replicative potential (due to telomere shortening) and an increase in the rate of senescent cell formation.

It's always helpful to link to papers you reference :-)


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Are cerebellar basket and stellate neurons actually different cell types?
The title more or less says it all, but to contextualise a bit:
Cerebellar molecular layer interneurons have been classified, probably since Cajal, into the basket cells, which synapse onto the soma and axon initial segment of Purkinje cells, and stellate cells, which synapse onto the dendrites. The types are distinguishable in tissue slices by positional and morphological differences, but it is at least arguable that the variation is continuous and the boundary somewhat arbitrary. Both types take basically the same inputs and deliver the same outputs, albeit in marginally different cellular locations.
As far as I'm aware -- and I would be incredibly happy to be told otherwise -- there is no known difference in protein expression that can be used to distinguish between these interneuron types, for instance in dissociated culture.
So, is there in fact some identifying marker? And if not, is it still useful to classify them as separate types?

You are completely right with your guess that is was Ramón y Cajal who first described these cells in his famous monograph "Histologie de systeme nerveux de l'homme et des vertebres." published in Paris Maloine in 1911.
Since the only method he had at his disposal was Golgi's staining which is a pure anatomical staining (there is no living cell left in the tissue after the treatment with  potassium dichromate and silver nitrate) with silver chromate filling completely single cells at random.
Using only this staining and his eyes Cajal did classify the neurons according to their size, location in the brain and their shape. This is why we have such names like basket, stellate, moss, pyramide etc. in neuron description. This classification is purely anatomical and does not reflect any intrinsic properties of these cell, as for example being motor neurons or interneurons.
When I was studying histology at my medical school I was always told that there is new "mollecular biology"-based classification of neurons is about to come and to replace the existing classification by Cajal, many cell types were supposed to be merged there, among others also different anatatomical groups of interneurons. (This was one of the major research topic of the research facility where I studied neurohistology).
As far as I can see, this classification is still not here...


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Why Does Salt Water Help Sore Throats?
I am having some trouble understanding how salt water, a simple solution, could so effectively remove the pains of a sore throat.
I do believe that the answer is closely related to hypo/hyper-tonic solutions, but why is this so, and how does this work?

Salt water may have anti-septic properties due to the effect it has on water potential.  Pure water has a water potential (Ψ) of zero.  A concentrated salt solution has a lower (more-negative) water potential.  The water potential of the salt solution is likely to be more negative than that of the pathogen's cytoplasm; the salt solution is therefore referred to as hypertonic.  Therefore water osmoses out of the cell (osmosis being the net movement of water from a higher water potential to a lower water potential across a semi-permeable membrane).  The loss of water from the pathogenic cells causes osmotic crenation - the cell becomes shrivelled and dies.
A hypotonic solution (for example cells placed into pure water) would cause the opposite effect - osmotic lysis.  This is the bursting of the cell due to the movement of water into the cell.  The bacterial cell wall would first have to be damaged (e.g. by penicillin).  This would not be the process by which a salt solution has effect, however.
The fact that the salt water is warm in order to improve solubility may also have the side-effect of causing vasodilation around the infection, increasing the rate at which white blood cells can arrive at the infection site.
It has been more difficult to find a theory as to why a salt solution would have analgesic properties, see the comments below & previous versions of this answer.

It is my understanding that a sore throat is sore because of inflammation. I had always thought that the analgesic effect of salt water was to reduce that inflammation by placing the inflamed tissue in a hypertonic solution. Do you have a reference that says that the salt water is actually killing the pathogenic cells or that this is the direct mechanism of action for why it "helps" the symptoms of sore throat?

It would be doubtful that any antiseptic effect as I described above would produce immediate "help" or pain relief. I agree that the solution would have anti-inflamtory effect, however as to the "sore throat being sore because of the inflammation" I must confess that I don't know.  If that is the case then of course that would be the direct mechanism that the op is looking for =)

Wouldn't it kill your own cells too and make the problem worse (at least temporarily?

Well it certainly wouldn't discriminate; I would propose that your own cells are less isolated, in that they will be near to a capillary with ready supply of water to replace any lost in the osmosis?

@Casebash It also helps to reduce inflammation by the same osmotic process - it draws the excess fluid around the cells thereby reducing the swelling. Reduced swelling would mean less irritation by the food/water entering the pharynx, as the contact surface is lessened.


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What are the major evolutionary pressures for Bioluminescence?
What are the major evolutionary pressures for Bioluminescence?

In which organisms? Certainly mushrooms, glowworms, and dinoflagellates illuminate for different reasons...

Sorry for not being specific. I was mostly curious about deep sea. Should I break this up into separate questions?

This question is close to my heart since I saw the Phosphorescent Bay in SW Puerto Rico. Its interesting to contrast the deep ocean with fresh water caves, where the fish become blind / eyeless.  The difference might simply be a lack of phosphorescent microorganisms to provide the light for the fish, or maybe the distant connection to the surface allowed the phosphorescent algae to influence the sunless deep parts of the ocean.

In many cases, and in particular in marine invertebrates, the bioluminescence is in fact produced by symbiotic bacteria of the Vibrionaceae family. In most cases the bacteria can sense when they are being hosted by the animal through quorum sensing mechanisms, and start producing light.
The evolutionary pressure for the bacteria to produce light is to be in the protected environment of the animal which often feeds them. On the animal side, there can be several reasons to host the luminescent bacteria. In many cases, the evolutionary pressure is linked to reproduction. In order to successfully reproduce in the immensity of oceans, marine animals need to know that they are among their kind before they release their gametes.
Another example is the one of some deep see fish like the anglerfish which have developed a special "flash light" organ to host bio-luminescent bacteria. Fish can use the bacteria to emit a light to aid in camouflage, hunting, and attracting mates. See http://microbewiki.kenyon.edu/index.php/Deep_sea_fish

Whilst it's not exactly academic, the BBC documentary "Blue Planet" had an excellent episode on deep-sea creatures, containing a large section about bioluminescence. Well worth a watch.

No matter the reason the "Blue Planet" is always worth watching.

@Polynomial In fact, the II came out about three years ago, but I haven't been able to see it yet.

Bioluminescence, in particular in marine organisms, has also been linked to way to get rid of reactive oxygen species (ROS). In fact, luciferases catalyse the photogenic oxydation of luciferins. In [1], the authors propose that this system's function was primarily for antioxydation, while in deep seas, where the water is oxygen-poor, the lower selective pressure toward antioxydation would have given rise to light emitting functions in specialized organs.
THE ORIGINS OF MARINE BIOLUMINESCENCE: TURNING OXYGEN DEFENCE
MECHANISMS INTO DEEP-SEA COMMUNICATION TOOLS
Bioluminescence in the Ocean: Origins of Biological, Chemical, and Ecological Diversity


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Are there any pre-Holocene venomous animals?
The novel (and subsequently movie) Jurassic Park featured a dinosaur called Dilophosaurus, that was purported to be venomous and had an ability similar to that of the extant spitting cobra. Unfortunately, the whole setup isn't real.
This got me wondering though. Modern venomous snakes, to give a particular example, ought to have had ancestors that already had appropriately modified salivary glands, and similarly for most other extant venomous species. (I suppose I'll restrict myself to vertebrates so as not to be too broad.)
I now ask: have fossils of animals that are likely to have been venomous ever been discovered?

Excluding invertebrates?

Squamates
Extant venomous snakes do have venomous ancestors. Fry et al. (2006) reported on finding venom toxins more broadly within Reptilia, beyond the well-known venomous snakes and the helodermatid lizards. They show that varanid and iguanid lizards also have venom toxins.
The same group of authors (Fry et al., 2009) then reported on the use of venom in the extant varanid lizard, Varanus komodoensis (Komodo monitor). This is a different hypothesis than the usual "toxic bacteria" hypothesis for predation in these animals. They then reinterpret the skull of the giant (~5.5 m) varanid Varanus (Megalania) priscus. They conclude, based on anatomical similarity and the close phylogenetic relationship between it and V. komodoensis, that V. priscus' predatory mode was more similar to other varanid lizards, likely involving venom delivery.
Archosaurs
Discovery of a venomous clade within squamates, including snakes and several clades of lizards, does not imply venom more broadly within Reptilia. Specifically, it would not parsimoniously reconstruct the presence of venom in Archosauria, the clade that includes dinosaurs. However, Gong et al. (2009) interpret the teeth of the dromaeosaurid theropod dinosaur, Sinornithosaurus as having venom grooves. These grooves are similar to grooves in the teeth of Uatchitodon, a Triassic squamate from Arizona.
There is currently no solid evidence that archosaurs could spit, nor is there ever likely to be.
Mammals
Venomous extant mammals include the platypus (Ornithorhynchus), Solenodon, and some species of shrews. The venom gland in the platypus is a modified sweat gland on the hind limb, which includes an ossified spur. A similar structure has also been found in the basal mammalian taxa Gobiconodon and Zhangheotherium (Hurum et al., 2006). Hurum et al. (2006) described a similar structure in the multituberculates. As the most basal clade of Mammalia, the authors conclude that an ossified spur is apomorphic for Mammalia and that the ancestral mammal may have used it for venom delivery. This structure would have been retained more-or-less unmodified in platypus and lost in other mammalian lineages. The authors' conclusions are supported by the presences of a non-venomous spur in the other clade of monotremes, the echidnas.
Fox and Scott (2005) report that the Palaeocene (~60 Ma) pantolestid Bisonalveus browni also possessed teeth with venom delivery grooves.
Non-gnathostomes
Szaniawski, 2009 used similar logic based on grooved "tooth" elements to conclude that some extinct jawless chordate conodonts may also have been venomous.

It always tickles me that the platypus is poisonous, despite looking so ridiculous.

@Polynomial Cough... they're venomous, not poisonous.


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Are single-celled organisms capable of learning?
I've read that the amoeba is capable of learning.  Since these protists have no nervous system, it's safe to assume that even highly simplified learning mechanisms of the Aplysia are miles off in the evolutionary distance.
How does this species learn?  I would presume that there is a complicated chain of receptor mediated transcription regulation, but what are the specifics?  What types of receptors does an amoeba have to sense its environs?

Amoebas are not animals, this term means wide range of similar, but not related unicellular organisms. Do you mean something like the learning behaviour of Physarum slime molde link?

Just a note: qualifying a species of "lower organism" could make some purist cringe ...
I dont have a straight answer, but you might be interested in this paper which describes a model of event memory in amoeba

@MartaCz-C Yes, I had written "animal" in and taken it out for a better substitute and ended up leaving it in.  You are absolutely correct about its misuse.  I have not encountered the study in your link, but I will take a look at it.

I'd like to know what is the reference for amoebic learning. I cannot comment directly on this, but there is some evidence for  "adaptive anticipation" in both prokaryotes and single-celled Eukaryotes which do not have a nervous system.
In the case of E. coli, it has been shown that the bacteria can anticipate the environment it is about to enter.  E. coli in the digestive tracts of mammals will typically be exposed to initially a lactose, and then later to a maltose environment as the bacteria pass down through the animal tract. This suggests that upon encountering a lactose environment, maltose operons are induced. I.e., upon encountering lactose, maltose is anticipated. This suggests a "genetic memory" of the sequence of sugar types where lactose is always encountered before maltose.
Further cultures (500 generations) of E. coli in the absence of maltose but in the presence of lactose reduced the maltose operon activity to negligible levels, suggesting that this is an adaptive prediction of environmental changes.
Mitchell, A et al., Adaptive Prediction of environmental changes by microorganisms, 2009, 460, 1038

That’s more like adaptation through evolution though. I don’t know about the OP, but when I’m thinking about “learning”, I usually think in terms of a single lifetime.

In addition to the excellent response up top (by Poshpaws), one can also imagine how these systems work by looking at recent synthetic examples of single-celled organism memory.
It is possible to design various bistable switches using protein pathways, RNAi, or other means that will latch a particular state. In that way, an organism could effectively "remember" one bit of data by querying the state of the toggle.
For a specific example, see the Gardner 2000 paper [1]. It's a transcription-level circuit, that in response to a certain stimulus can latch either high or low. While the synthetic version itself is not terribly robust, one could see how in nature a highly evolved / refined circuit could maintain state and effectively serve as "memory" for a single-celled organism.
[1] Gardner, et. al 2000 "Construction of a genetic toggle switch in Escherichia coli."

This is an interesting point. A big issue with the toggle switch is that the network topology from Gardner's paper is rarely if ever seen in biology. However, there remains plenty of examples where hysteresis may occur in naturally relevant systems.

The authors of The omnistat: A flexible continuous‐culture system for prolonged experimental evolution report on an experiment where they evolved Lactococcus lactis to have an anticipatory response to their environment. A culture was set up in their bioreactor, and periodically one of two weak bases was introduced: either lactate or acetate, and then, depending on the base, fresh media of fructose or galactose was introduced. The simple idea is depicted below:
1. randomly choose either:
  dose with dilute acetate -> fructose comes next
  dose with dilute lactate -> galactose comes next
2. go back to step 1.

The culture is continually exposed to this treatment over many days or weeks (it's not clear from their paper), during which, there is an evolutionary pressure for the bacteria to engineer a response between the weak base and future energy source. That is, a bacterium that can shift its metabolism slightly towards processing fructose faster when acetate is present will have higher fitness, and likewise of lactate/galactose.
In the end, after many rounds, the bacteria have encoded the relationship into their genes - a type of "learning" that the other answers to this question also give examples of.


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Where does vasoconstriction occur in limbs?
When cold or in shock a person's blood vessels constrict in order to preserve heat or to move blood flow to protect vital organs.  I am interested in the mechanism by which this is achieved.  Does this occur along the entire length of the limb or are there 'pinch points' (for example is it possible to simply have a point of vasoconstriction just below the shoulder which would reduce blood flow to the entire arm)?  Am I correct in thinking that the response is neurological rather than endocrinological ?

Reduced blood flow to a region of the body occurs through 2 principal mechanisms.
1) The smooth muscle fibers in the tunica media layer of the arteries contract and reduce the diameter of the artery, limiting blood flow due to increased resistance (this is the mechanism in @agrimaldi's answer).
2) Circularly oriented smooth muscle fibers at the junction of a metarteriole (the distal end of an arteriole) and a capillary bed form a precapillary sphincter, which serves as a valve and prevents blood flow into a the capillary bed.
So to answer your question, it is both.  The narrowing of the arteries would occur rather continuously across the limb but there are "pinch points" where metarterioles joint the capillary beds.
The endocrine and nervous systems are pretty integrated so in most cases it would be the result of the actions of both systems.

So it would be possible to, for example, have one hand receiving less blood flow than the other?

I would think so, yes.  You could likely test this by placing one hand in cold water and the other in hot water.  The blood vessels in the hand in cold water should constrict and the ones in the hot water should dilate. (I think)

Vasoconstriction is a phenomenon that can be caused by many factors, from mechanical events (such as stretching) to hormones (epinephrin, neurepinephrin, angiotensin ...). But no matter what is the cause, the end-result is an increase in intracellular calcium concentration in smooth muscle cells (which compose the arteries and veins). So it seems that constriction does not happen in specific points but rather all along the limb. Furthermore angiotensin receptors are found in high concentration in smooth muscle cells [1], suggesting that every such cell has the ability to change it's morphology in order to induce vasoconstriction.
As for your second point, I think the response is as much endocrinological as neurological, since it involves both peripheral and central regulations. (Well, as a side note, endocrine systems rarely have no interaction with central ones).
Angiotensin receptors : distribution,
signalling and function

"endocrine systems rarely have no interaction with central ones" - +1 just for this.


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Substrates of cytochemical reactions in this immunostaining
Expression of extracellular protein Laminin 9 alpha-4 chain in human skeletal muscle.
Indirect immunostaining with HRP immunostain marker. Ob.x40.
I have unsuccessfully searched NCBI -database, JSTOR and other major Biology databases for an answer. This suggests to me that I do not understand what is going on.
1. What are the substrates of above cytochemical reactions for the given immunostaining above?

I need to give a simpler question where I apparently know the reaction exactly, since it is possible that people cannot answer the above question.
Mitochondria in Hep-2 cell line cells. Cytochemical test for mitochondria specific enzyme NAHD dehydrogenase. Ob.x40.
2. What is the substrate of the cytochemical reaction?
My answer:
The reaction of NADH dehydrogenase is:

NADH + H+ + CoQ → NAD+ + CoQH2

Substrate: CoQ

I'm having a hard time understanding this, it looks like some context is missing. If you're posting homework, please provide the full text of the problem and show what you've tried so far and what exactly you don't understand.

@MadScientist: Please, see pages 3 and 4 of the file: http://dl.dropbox.com/u/175564/Practical%20works%202011-2012.pdf

For my benefit can I ask why the in and out are included in the equation?

@Rory Because I was stupid when copy-pasting the given equation without deleting them, since we do not need them in my opinion. Right?

@Rory I removed 4H from the both sides. Correct now?

@Masi Looks fine :)

It's hard to understand the question, but in any immunocytochemical staining such as the above, you have two different types of reactions:

the antibody binding to the target (in this case, some laminin)
the peroxidase-based colorimetric reaction with DAB. DAB (3,3'-diaminobenzidine tetrahydrochloride) is oxidized in the presence of hydrogen peroxide to form a brown precipitate, which becomes the stain.

The fact that is an indirect immunostaining implies that you have a non-conjugated primary antibody against laminin and a secondary antibody against the Fc part of immunoglobulins of the species in which the first antibody was raised. This secondary antibody will be conjugated with HRP (horseradish peroxidase, the enzyme responsible of generating the peroxides that will oxidize the DAB).
For a more complete reference of immunocytochemical stainings, you can read: http://www.ihcworld.com/_books/Dako_Handbook.pdf

Thank you for your answer! Need to read the book in my Christmas holiday.


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Mechanism of syndesmophyte growth in AS
Ankylosing Spondylitis (AS) causes inflammation around joints and the growth of syndesmophytes that may eventually fuse vertebrae. I'm familiar with the genetics (HLA-B27, IL1A) related to the condition, but I can't find any information about the mechanism that causes the actual growths to occur.
My current assumption is that AS causes the over-production or under-production of a particular compound or enzyme at the growth site, but I can't find any studies or papers that explain this. Is the mechanism known? Is it directly related to abnormal levels of a particular substance?

As far as I remember the pathology course from medical school, chronic long-lasting inflammation often leads to proliferation of connective tussie and ultimately to fibrosis.
The actual mechanism here is the lack of oxygen which is used-up by different immune system cells to produce peroxydes and superoxydes.

Interesting. So the symptomatic inflammation over long periods of time ultimately causes fibrosis and fuses the vertebrae? Also, what causes the lack of oxygen at the site?

Not only symptomatoc, any inflammation (including the idiopatic one) would do. There are many reasons for hypoxie here: decreased blood flow rate, swelling etc.


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What are the criteria for determining the influence of epigenetic factors?
Isolating a gene or sets of genes in diseases sometimes isn't enough to determine penetrance - epigenetic factors can have a significant effect. What are the criteria in determining whether epigenetic factors are significant?

First of all, the nature of penetrance is almost entirely unknown.  Likely it's a combination of epistasis and gene interactions, induced gene regulatory pathways, developmental noise, and other factors.  Epigenetics (imprinting, etc) may have little to do with penetrance, while chromatin structure may be a consequence of other things (most now regard histone modifications, etc, as consequence of transcription rather than heritable regulatory mechanism).  Currently, the field of epigenetics is undergoing a (long-overdue) reassessment.  Until that happens, anyone who wants to make claims of "epigenetics" is free to, so spurious claims are rampant.

Do you know any relevant papers/reviews on the issue?

Sorry, reviews on which?  Penetrance, epigenetics, or chromatin structure?

Sorry, my comment was not clear. I was wondering if there were reviews on the current view of epigenetics. You are saying that the field needs a reassessment, I was wondering if someone had analysed this issue in detail (why it need it, what are the things to better define etc)

A few years ago Youngson and Whitelaw wrote a review that started to separate epigenetics from inducible gene expression (http://chd.ucsd.edu/_files/winter2009/Youngson.TransgenerEpigenEffects.08.pdf), but it didn't go far enough.  There have been studies lately that show that histone modifications are probably consequence of gene expression, rather than carrying memory.  Similarly there are studies that show that DNA methylation does not "shut off" expression, and there is still not a single case showing that methylation changes alter endogenous gene expression.

Thanks, that is a good start. So, correct me if I am wrong, you are saying that it has been been shown that the methylation status can change depending on gene transcription, but not that methylation modulates transcription?

I don't know how kosher it is to redirect within the stack, but here is this: http://biology.stackexchange.com/questions/391/how-do-cpg-islands-remain-unmethylated/392#392

Yep, I read your other reply :) Thanks a lot.


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Do plants produce any heat?
Many plants (e.g. roses, palms) can be protected from frost during the winter if shielded with an appropriate coat that can be bought in garden shops. Do plants produce any heat that can be kept inside with these "clothes"?

Some produce quite a lot, like Skunk Cabbage and the Voodoo Lily. The latter can even sustain temperatures over 15 degrees celsius above its surroundings. This is a fairly good article on exothermic plants

The senator tree in Florida produced heat. It burned to the ground from internal combustion.

Cellular respiration in plants is slightly different than in other eukaryotes because the electron transport chain contains an additional enzyme called Alternative Oxidase (AOX). AOX takes some electrons out of the pathway prematurely - basically the energy is used to generate heat instead of ATP.
The exact purpose of AOX in plants is still unclear. Plants will make more AOX in response to cold, wounding, and oxidative stress. We know of at least one plant (skunk cabbage) that exploits this pathway to generate enough heat to melt snow. This link gives a pretty good overview.

(AOX is dear to my heart, since my first 3 years working in a laboratory were spent studying this gene <3)

This is interesting @Amy. Why do you say that the exact purpose of AOX is unclear? Do you mean it is unclear what AOX does, or it is unclear why it does it?

@Amy The melting snow part is really fascinating, +1

@poshpaws what AOX does is pretty well understood, but the "why" isn't completely clear. When the electron transport chain converts energy into heat, it's "lost" rather than doing something useful - making ATP. It may be because since plants can't run away from their problems (temperature, wounding from bugs, and other things that lead to reactive oxygen species) they need an additional way to deal with oxidative stress that other organisms never developed.

I always thought the purpose of AOX (apart from heating) was as an electron sink to help decrease photoinhibition, in the same way that photorespiration is.

This respiration pathway also called cyanide resistant respiration!

Plants will be respiring continuously, which is an exothermic process. Therefore the plants will be producing a small amount of heat. The protection from frost may be more a result of the vastly smaller convection current of the coat compared to the atmosphere rather than by reducing any conduction away of heat produced by the plant, however.
Keeping the plant out of the wind by 'dressing it' will reduce the rate of transpiration when the stomata are open.  I would very tentatively suggest that, as water has a very high specific heat capacity, having a greater volume of water within the plant would help to retain any heat that was produced by respiration.  However this is entirely speculation on my part.

Snow is also a good insulator, and these "coats" may mirror that effect. See here for some measurements of the temperature difference: http://snailstales.blogspot.com/2010/02/temperature-under-snow-part-3.html

"Snow is also a good insulator" - indeed, that's how igloos work.

Some plants have mitochondrial pathways that produce heat. That's why skunk cabbage can be seen poking outing of the snow in late winter/early spring. Here's a review:  http://onlinelibrary.wiley.com/doi/10.1111/j.1744-7909.2010.01004.x/pdf

These and other plants make use of the cyanide-resistant pathway. This is a short-cut in the electron transport chain that allows the plant to produce extra heat at the expense of not using those electrons to manufacture ATP. (It is called the cyanide-resistant pathway because it provides an alternate pathway for the electrons to take. Rather than killing the cell quickly via free-radical production, it is slowly starved via lack of ATP.)

Yes, respiration is exothermic and plants (like all living things) respire.
I think the effect of covering the plant has to more to do with reducing sensible heat loss , e.g. direct transfer of heat from plant to air) rather than latent heat loss (through evaporation).
The cover is effectively reducing the mixing of the air near the plant (and closer to the temperature of the surface of the earth) with the free atmosphere (closer to the temperature reported by the weatherman)


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How are the boundaries of a gene determined?
What statistical processes and methods are used by geneticists/molecular biologists to know where one gene starts and one ends?

What does the "basic" tag refer to?

I thought that the question was more of a foundational one to seed the group, so I tagged it basic . If that's not protocol, please feel free to remove.

@ghchinoy: I retagged it, assuming it's a meta tag (although it is a question about base pairs)

Just to clarify: we’re talking about protein-coding genes here, right? There are many more for which the methods are completely different.

@KonradRudolph could you perhaps reference the other gene types and methods? Thanks.

@ghchinoy Just as an example, I’m currently working on tRNA genes and since they’re using a different polymerase their promoter and termination site look markedly different. The same is true for all the other non-coding RNAs and then there are things like pseudogenes and LINEs/SINEs (those are not usually considered genes but due to their similarity to non-coding RNA genes they complicate the analysis). Still, there actually are bioinformatical methods to find those genes. They predominantly use motif search as far as I know.

I know of only one naive approach to determining the boundaries of a gene : RACE-PCR. There are two kinds, 3' and 5' RACE, which allow to find the respective extremities.
The rationale is the following :

You perform a reverse transcription of the transcript of interest using a specific primer. At this step you have a specific single stranded cDNA.
Then you add a stretch of identical nucleotides called the homopolymeric tail in 5' of the cDNA.
Finally you perform a PCR using one specific primer and one universal primer that recognizes the homopolymeric tail. You can sequence your amplified cDNA and find where it is located in the genome with a 1 bp resolution.

For the 3'RACE, the concept is the same but the poly-A tail is used instead of generating it yourself with the terminal transferase.
See this paper for a detailed protocol :
 Sambrook J, Russell DW. 2006. Rapid Amplification of 5’ cDNA Ends (5'-RACE). CSH protocols 2006.
Also, the corresponding wikipedia article gives you more details about what is happening at each step, but beware, there is an error : it is said that for the 5'RACE, the terminal transferase appends the homopolymeric tail in 3' while it appends it in 5'

-1: that may be a good approach to see the boundaries of an ORF (to be honest, you don't always need a RACE, a simple PCR may work as well), not of a gene. What about promoter and regulatory elements? Also, what is the advantage compared, say, to a bioinformatics approach after sequencing?

@nico : so, with the definition you provide, a gene has no boundaries.

@nico : Ok, I see your point, but I don't think the OP had this definition of a gene in mind. Also, I totally agree that new technologies such as RNA-seq give you a more comprehensive answer for genome annotation.

we can discuss for hours about the correct definition of a gene, but I don't think there is much to discuss about the fact that the promoter is part of a gene. And by retrotranscription of mRNA you don't get the promoter.

There are various software in which you can enter your sequence (let's say the whole genome sequence) and it can identify for you the putative open reading frames (ORFs), i.e. the start codons and the stop codons. Then, by using these putative genes, you can do a sequence alignment by using BLAST and then, based on the scores you can confirm that those are really ORFs. As this being the statistical approach, you can then verify your results in the wet lab, like agrimaldi suggested.

But how do those software determine gene boundaries? What are they looking for that is indicative of a gene boundary?

Maybe another question should be started specifically about what programmatic techniques are used? Maybe with a bioinformatics tag.

@RichardSmith They are basically looking for start codons (ATG, GTG) that define the beginning of the open reading frame (ORF), and stop codons (TAG, TAA, TGA), that define the end of the ORF, and also checks whether the number of bases between the start codon and the stop codon is devisible by 3.

@ghchinoy Yes, that could be interesting, but I don't think it is more complicated than I already explained to Richard. You can of course add a few more "checks" the software can do, like the length of the ORF.

If your goal is to define the boundaries of the transcription unit (the part of the DNA that is transcribed) the above answer is accurate, although many people merely use homology to cloned cDNAs rather than RACE reactions.  This approach has the benefit of defining alternative splice forms at the same time.
If your goal is to define the "ends" of the gene, it can only be done empirically and functionally because control elements (boundaries, enhancers, etc) are impossible to recognize using informatics, and even if one finds enhancers, it's not certain that those enhancers are used with specific genes.  Some genes can be millions of base pairs long, so have hundreds of others genes interspersed.  The "gold standard" for defining the boundaries of genes is to rescue the loss-of-function phenotype of a mutation with a transgene that contains the gene of interest.  If the DNA that is transformed back into an organism can recover the wild-type condition of a mutation of a gene, it is assumed that all of the important parts of that gene are within the transgene.

Generally speaking you sequence the genome and then search for clues. There are usually specific sequences preceding a gene that help the translational equipment know "hello this is where we begin" as well as regions where proteins can bind that are used to enhance or inhibit translation of the gene.
Computers can be programmed to search through the sequence and bring up possible candidates for people to look at more closely.


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How is duration of efficacy estimated for vaccines?
Vaccines, especially those given in adulthood, usually have term limits attached, eg: 10 years for yellow fever or 3 years for typhoid. Since presumably the time course of an immune response is no great respecter of our calendrical conventions, and since there is also presumably a spectrum of responses across the population, how are these durations estimated? What are the criteria for deciding a cutoff time?
Also, do the estimates get revisited and updated as time goes on and new data become available? Do new data become available? Is there continuing follow-up research into this, or does the estimate just get made before the vaccine comes to market and then get taken as gospel?

Duration of efficacy is typically determined by tracking the antibody titers of a cohort of subjects who have gotten the vaccine, and estimating based on the trajectory of those titers where they will eventually cross the threshold to the point where the vaccine no longer confers immune resistance.
These estimates do get revised and estimated as time goes on - you will occasionally see new recommendations for a second or third "booster" dose of a vaccine, which is meant to extend the duration of immunity beyond the duration of the original vaccine.
Two major types of studies track this over time. Phase IV clinical trials, which are clinical trials required post-licensure, and observational epidemiology trials, which tend to be performed when disease transmission starts to occur in supposedly vaccinated populations.

Mechanical snail has raised the issue of viral evolution, so I'll touch on it briefly. The duration of efficacy discussed above is based on how long a patient's body can mount an immune response to a particular challenge. That's a concern for all vaccines.
For some vaccines, there's a secondary process that's of concern - that of the virus evolving in such a way that the antigens targeted by the vaccine are no longer those on the virus itself. This is only a concern for some viruses, notably those that are particularly fast-evolving, like influenza or HIV, and less of an issue for say, measles and HPV.
But that's typically not what people are talking about when they say "duration of efficacy" because it's inherently unpredictable, and less a function of the vaccine and more a function of the virus.

Is the cutoff determined by the first subject with titer below the threshold, the last or by some percentile of them (eg. 50)?

Usually with some form of statistical model

I read somewhere (I don't have a citation) that duration of efficacy is limited by the evolution of viral antigens; eventually the population will evolve to the point where the immune system no longer recognizes them effectively. RNA viruses have more volatile genomes and so their antigens drift faster. This explanation makes some sense but it conflicts with @Epigrad's answer.

Community wiki because I'm not sure this is correct. Feel free to edit in more information.

I've added some details to my answer. Your answer doesn't particularly conflict, its just asking about a subtly different process.

authors: w̢in̡͢g͘̕ed̨p̢͟a͞nt̡͜͝he̸r̴


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Is there any evidence that sexual selection may lead to extinction of species?
Darwin suggested that sexual selection, especially by female choice, may counter natural selection. Theoretical models, such as a Fisherian runaway process, suggest that evolution of preference and preferred phenotypes may drive each other in ever increasing speed.
Because one male may fertilize many females, one could imagine that natural selection against preferred but energetically costly phenotypes may be weak, and the whole process may not slow down fast enough (i.e., be sufficiently self-limiting). If male mortality is high and their number is low, the random fluctuations may easily cause the extinction of population.
Is there any fossil or experimental evidence that this may really happen?

TL;DR:

There is a dearth of actual experimental evidence. However:

there is at least one study that confirmed the process ([STUDY #7] - Myxococcus xanthus; by Fiegna and Velicer, 2003).
Another study experimentally confirmed higher extinction risk as well ([STUDY #8] - Paul F. Doherty's study of dimorphic bird species an [STUDY #9] - Denson K. McLain).

Theoretical studies produce somewhat unsettled results - some models support the evolutionary suicide and some models do not - the major difference seems to be variability of environmental pressures.
Also, if you include human predation based solely on sexually selected trait, examples definitely exist, e.g. Arabian Oryx

First of all, this may be cheating but one example is the extinction because a predator species specifically selects the species because of selected-for feature.
The most obvious case is when the predator species is human. As a random example, Arabian Oryx was nearly hunted to extinction specifically because of their horns.

Please note that this is NOT a simple question - for example, the often-cited in unscientific literature example of Irish Elk that supposedly went extinct due to its antler size may not be a good crystal-clear example. For a very thorough analysis, see: "Sexy to die for? Sexual selection and risk of extinction" by Hanna Kokko and Robert Brooks, Ann. Zool. Fennici 40: 207-219. [STUDY #1]
They specifically find that evolutionary "suicide" is unlikely in deterministic environments, at least if the costs of the feature are borne by the individual organism itself.
Another study resulting in a negative result was "Sexual selection and the risk of extinction in mammals", Edward H. Morrow and Claudia Fricke; The Royal Society Proceedings: Biological Sciences, Published online 4 November 2004, pp 2395-2401 [STUDY #2]

The aim of this study was therefore to examine whether the level of
  sexual selection (measured as residual testes mass and sexual size dimorphism) was related to the risk of extinction that mammals are currently experiencing. We found no evidence for a relationship between these factors, although our analyses may have been confounded by the possible dominating effect of contemporary anthropogenic factors.

However, if one takes into consideration changes in the environment, the extinction becomes theoretically possible. From "Runaway Evolution to Self-Extinction Under Asymmetrical Competition" - Hiroyuki Matsuda and Peter A. Abrams; Evolution Vol. 48, No. 6 (Dec., 1994), pp. 1764-1772: [STUDY #3]

We show that purely intraspecific competition can cause evolution of extreme competitive abilities that ultimately result in extinction, without any influence from other species. The only change in the model required for this outcome is the assumption of a nonnormal distribution of resources of different sizes measured on a logarithmic scale. This suggests that taxon cycles, if they exist, may be driven by within- rather than between-species competition. Self-extinction does not occur when the advantage conferred by a large value of the competitive trait (e.g., size) is relatively small, or when the carrying capacity decreases at a comparatively rapid rate with increases in trait value. The evidence regarding these assumptions is discussed. The results suggest a need for more data on resource distributions and size-advantage in order to understand the evolution of competitive traits such as body size.

As far as supporting evidence, some studies are listed in "Can adaptation lead to extinction?" by Daniel J. Rankin and Andre´s Lo´pez-Sepulcre, OICOS 111:3 (2005). [STUDY #4]
They cite 3:

The first example is a study on the Japanese medaka
  fish Oryzias latipes (Muir and Howard 1999 - [STUDY #5]). Transgenic males which had been modified to include a salmon growth-hormone gene are larger than their wild-type counterparts, although their offspring have a lower fecundity (Muir and Howard 1999). Females
  prefer to mate with larger males, giving the larger
  transgenic males a fitness advantage over wild-type
  males. However, offspring produced with transgenic
  males have a lower fecundity, and hence average female
  fecundity will decrease. As long as females preferentially
  mate with larger males, the population density will
  decline. Models of this system have predicted that, if
  the transgenic fish were released into a wild-type
  population, the transgene would spread due to its mating
  advantage over wild-type males, and the population
  would become go extinct (Muir and Howard 1999).
  A recent extension of the model has shown that
  alternative mating tactics by wild-type males could
  reduce the rate of transgene spread, but that this is still
  not sufficient to prevent population extinction (Howard
  et al. 2004). Although evolutionary suicide was predicted
  from extrapolation, rather than observed in nature, this
  constitutes the first study making such a prediction from
  empirical data.
In cod, Gadus morhua, the commercial fishing of large
  individuals has resulted in selection towards earlier
  maturation and smaller body sizes (Conover and Munch
  2002 [STUDY #6]). Under exploitation, high mortality decreases the
  benefits of delayed maturation. As a result of this,
  smaller adults, which mature faster, have a higher fitness
  relative to their larger, slow maturing counterparts
  (Olsen et al. 2004). Despite being more successful
  relative to slow maturing individuals, the fast-maturing
  adults produce fewer offspring, on average. This adaptation,
  driven by the selective pressure imposed by
  harvesting, seems to have pre-empted a fishery collapse
  off the Atlantic coast of Canada (Olsen et al. 2004). As
  the cod evolved to be fast-maturing, population size was
  gradually reduced until it became inviable and vulnerable
  to stochastic processes.
The only strictly experimental evidence for evolutionary
  suicide comes from microbiology. In the social
  bacterium Myxococcus xanthus individuals can develop
  cooperatively into complex fruiting structures (Fiegna
  and Velicer 2003 - [STUDY #7]). Individuals in the fruiting body are
  then released as spores to form new colonies. Artificially
  selected cheater strains produce a higher number of
  spores than wild types. These cheaters were found to
  invade wild-type strains, eventually causing extinction of
  the entire population (Fiegna and Velicer 2003). The
  cheaters invade the wild-type population because they
  have a higher relative fitness, but as they spread through
  the population, they decrease the overall density, thus
  driving themselves and the population in which they
  reside, to extinction.

Another experimental study was "Sexual selection affects local extinction and turnover
in bird communities" - Paul F. Doherty, Jr., Gabriele Sorci, et al; 5858–5862 PNAS  May 13, 2003 vol. 100 no. 10 [STUDY #8]

Populations under strong sexual selection experience
  a number of costs ranging from increased predation and
  parasitism to enhanced sensitivity to environmental and demographic
  stochasticity. These findings have led to the prediction that
  local extinction rates should be higher for speciespopulations
  with intense sexual selection. We tested this prediction by analyzing
  the dynamics of natural bird communities at a continental
  scale over a period of 21 years (1975–1996), using relevant statistical
  tools. In agreement with the theoretical prediction, we found
  that sexual selection increased risks of local extinction (dichromatic
  birds had on average a 23% higher local extinction rate than
  monochromatic species). However, despite higher local extinction
  probabilities, the number of dichromatic species did not decrease
  over the period considered in this study. This pattern was caused
  by higher local turnover rates of dichromatic species, resulting in
  relatively stable communities for both groups of species. Our
  results suggest that these communities function as metacommunities,
  with frequent local extinctions followed by colonization.

This result is similar to another bird-centered study: Sexual Selection and the Risk of Extinction of Introduced Birds on Oceanic Islands": Denson K. McLain, Michael P. Moulton and Todd P. Redfearn. OICOS Vol. 74, No. 1 (Oct., 1995), pp. 27-34 [STUDY #9]

We test the hypothesis that response to sexual selection increases the risk of extinction by examining the fate of plumage-monomorphic versus plumage-dimorphic bird species introduced to the tropical islands of Oahu and Tahiti. We assume that plumage dimorphism is a response to sexual selection and we assume that the males of plumage-dimorphic species experience stronger sexual selection pressures than males of monomorphic species. On Oahu, the extinction rate for dimorphic species, 59%, is significantly greater than for monomorphic species, 23%. On Tahiti, only 7% of the introduced dimorphic species have persisted compared to 22% for the introduced monomorphic species.
...
Plumage is significantly associated with increased risk of extinction for passerids but insignificantly associated for fringillids. Thus, the hypothesis that response to sexual selection increases the risk of extinction is supported for passerids and for the data set as a whole. The probability of extinction was correlated with the number of species already introduced. Thus, species that have responded to sexual selection may be poorer interspecific competitors when their communities contain many other species.

Many thanks for all articles! I'll take a look on it (though it would take some time).

This answer is an inspiration to everyone on all of Stack Exchange.  Thank you!

Relating to your last comment on random fluctuations in survival, a recent theoretical paper by Lee et al. 2011 studies the effect of mating systems on demographic stochasticity in small population. No empirical data there though. Their main conclusion is that polygyny (in relation with sex ratio) can lead to high demographic variance, therefore lowering stochastic population growth rate and increasing extinction risk. The general effect is known as demographic stochasticity, a sampling effect leading to random variation in realized demographic rates due to small population size.


text:
How many people's DNA were involved in the compilation of the reference human genome?
I know that the reference human genome is complied from DNA portions from different people, most of whom were European. Do you know how many of them were involved? Do you know which ones were involved? Perhaps scientists like James Watson and George Church's DNA were in the reference genome. Also, I would assume that the NIH funded Human Genome Project's reference genome must be slightly different from the privately funded one.

tl;dr: In the Human Genome Project, they used the DNA of four people (though one male provided >70% of DNA. The Celera genome was compiled from five people.

In the [...] Human Genome Project (HGP), [...] scientists used white
  blood cells from the blood of two male and two female donors (randomly
  selected from 20 of each) -- each donor yielding a separate DNA
  library. One of these libraries (RP11 [anonymous donor from Buffalo,
  NY]) was used considerably more than others, due to quality
  considerations.
[...]
In the Celera Genomics private-sector project, DNA from five different
  individuals were used for sequencing. The lead scientist of Celera
  Genomics at that time, Craig Venter, later acknowledged (in a public
  letter to the journal Science) that his DNA was one of 21 samples in
  the pool, five of which were selected for use.
On September 4, 2007, a team led by Craig Venter published his
  complete DNA sequence,[21] unveiling the six-billion-nucleotide genome
  of a single individual for the first time.)

Source: Wikipedia

Can you please give me a link to the source of this text?

@GerganaVandova: Please follow the Wikipedia link. If you want original references, have a look at References 17-21.

Wow, I read so many papers regarding this matter, but never wikipedia. Thanks. But do you think it is really unknown exactly from which source the DNA came?

@GerganaVandova: If the HGP can track down RP11, or any other of the chosen donors, then they did a poor job at anonymizing the donors and might be sued for that. With Celera, who knows who might know? I'd almost bet that Venter was one of the five; I guess it may be possible to try and find out by aligning the Celera genome to the Venter genome.

Haha, is Venter's genome public?

@GerganaVandova: Yes, it is

I don't understand the point of this thread. The question was asked, and the accepted answer is just lifted from wikipedia. Why not just look in wikipedia in the first place? Is this the purpose of SE?

@Poshpaws: You may want to bring that up on Meta.

@Jonas Will do :)


text:
Last-ditch efforts to maintain thermal homeostasis
I was in the gym's steam-room today and a thought occurred to me: have I truly thwarted all possible mechanisms for maintaining thermal homeostasis?
There's sweating, which is thwarted because the steam-room's atmosphere is as close to 100% humidity as possible, so there's almost no evaporative cooling.
There's convection, which is thwarted because the ambient temperature is above normal body temperature.
And I can't get rid of heat by exhaling, because every lungful of air I inhale is already above normal body temperature.
I think that, eventually, I should go into hyperthermia, but beside the above, are there any other last-ditch attempts to lower core temperature that my body could take?

Thermotaxis? :)

Another factor at play here is the cardiovascular system.  Biro, Masudo, et al (2003) wrote an article about the benefits of the induction of systemic vasodilation for those with congestive heart failure.  This may be a short-term adaptation of the body stemming from the reduced ability to sweat in those conditions, or it may be a harbinger of the more serious circulation changes below.
Epstein and Roberts (2011) explain how the eventual results of heatstroke (which isn't the phenomenon that you are describing, but a sauna might induce some of the features of these same pathways) can cause a circulatory collapse due to an initial overtaxing of the circulatory system to compensate as well as the activation of certain heat shock proteins.
There are a lot of other references out there on sauna physiology, but many are not in evidence-based journals, so you can make your own decisions on those.
One factor that I didn't find mentioned explicitly was the basal metabolic rate.  It's probably safe to assume that the body will limit its own processes and reactions which produce heat.  I'm not clear on the extent to which factors such as the level of interleukins, etc. would play a role as they do in fever regulation.


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How does the sensitive plant detect vibrations?
The sensitive plant (Mimosa pudica) is a remarkable little plant whose characteristic feature is its ability to droop its leaves when disturbed:

Apparently, this ability to droop rests on the cells in the leaves of the sensitive plant being able to draw water out of themselves through changes in intracellular ion concentrations, which makes the leaves less turgid.
What I'm hazy about is how the plant "senses" vibrations. Plants don't really have a nervous system to speak of; how then does the sensitive plant "know" to droop when disturbed?

The leaves are not actually drooping, they are pivoting upwards. Drooping would be easier, but would not perform the crucial function of the closure, which is to reduce evapotranspiration from the stomata which are mostly on the upper (adaxial) leaf surface.

In fact, the idea of a plant nervous system is quite serious and constantly developing; of course those are rather local, simple signal pathways rather than an "animalian" centralized global network, but they use similar mechanisms -- depolarisation waves, neurotransmitter-like compounds, specialized cells... Here is a review paper by Brenner et al.
In the case of Mimosa, there is a good paper summing up Takao Sibaoka's long research of the topic.
In short, it seems that its petioles' phloem has cells which have polarized membranes and can trigger depolarization due to a mechanical stimulation. The signal then propagates to the corresponding pulvinus by a mixture of electrical and Cl- depolarization waves.
In the pulvinus, this signal triggers a second depolarization which coordinates the pulvinus' cells to trigger water pumping responsible for the leaf drop.
The transmission to the adjacent leaves is most likely mechanical, i.e. the movement of one dropping leaf excites another.
References:

 Brenner ED, Stahlberg R, Mancuso S, Vivanco J, Baluska F, Van Volkenburgh E. 2006. Plant neurobiology: an integrated view of plant signaling. Trends in plant science 11: 413–9.
 Sibaoka T. 1991. Rapid plant movements triggered by action potentials. The Botanical Magazine Tokyo 104: 73–95.

Haswell, Peyronnet, et al (2008) have shown that plants have some of the same mechanosensitive ion channels that E. coli does.  The work was on the roots of Arabidopsis and not the plant you are investigating, but the mechanism could be similar.
It would not seem that vibration plays a role, as it the plant seems responsive to the touch of the human rather than his/her approach to the leaf. This could rule out air currents as a mechanism (but J.M. has noted in the comments that they do fold up when you blow on them).

I wouldn't entirely rule air currents out. There are some of these plants growing in my backyard, and they do fold up when blown on.

@J.M. I'll certainly take your word for it!  Though, it could be that blowing on it is bending the leaf and still activating the mechanoreceptor.

Some additional notes- I have grown TickleMe Plants (sensitive plants) for years and  I have seen the plants close their leaves as a reaction to the vibrations caused by my foot steps as well as blowing on the plant and placing the plant in cold, very hot and dark environments.  In addition I have found the plant's ablity to detect vibrations I have observed to be dependent on temperature and the time of day. Some of this information can be found in The TickleMe Plant Book found at http://www.ticklemeplant.com  I am unable to specifically say what the mechanism is that detects the vibrations and causes the TickleMe Plants turgor pressure to change. I look forward to more answers.


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Which aspects of renal physiology are standing in the way of an artificial (mechanical) kidney?
Scientists have been able to create artificial organs with varying degrees of success.  The mechanical heart (in its various forms, e.g. ventricular assist) is able to sustain life for some period of time.
Efforts to grow whole organs in the lab will probably ultimately lead to a more pragmatic solution.  What aspects of the renal physiology are standing in the way of an implantable mechanical kidney (fashioned more so from tubules and membranes than simply being a shrunken down dialysis machine)?

Have you encountered Shuvo Roy's work?

@J.M. No, I had not previously.  I had looked into this about 10 years ago, but it looks like the field has come pretty far.  (I'll pull papers, but) Do they actually have a deliverable?

Apart from press releases, I don't think I've seen the release of a device for public consumption. Still, they're reporting results with the prototype. It's a step forward, yes?

@J.M. It's definitely a huge step forward.

The problem is that real organs are just damn complex - yes the kidney's prime role is just to be a filter, but in order to do so it must be plugged in to a dozen regulation mechanisms - osmotic balance, ion management, protein management and a plethora of more subtle ones. Moreover it is a part of body, so it must also follow all the standard protocols to live with immune system, obtain necessary resources to its function and maintenance, cooperate with nearby tissues...
Currently we only have rough knowledge about major processes, deciphering them all is a work for many, many years (if it is not futile at all).  Finally, our technology will be long not capable of implementing all those protocols; in peaks of perfection we can serially do simple parts in 100nm scale (microprocessors), while this is a scale of a complete molecular device.

I question your "100nm scale microprocessors" remark. We can manufacture microprocessors down to at least 30nm, if not lower. I think the current minimum transistor size is 14nm. Update: Intel and IBM are using 14nm in next-gen fabrication, several other companies are using 15nm. Intel have also been experimenting with 12nm and 11nm units.

Note that the above just refers to the overall transistor size. The internal layers and interconnects are often smaller than 1nm in at least one dimension. Clearly we already have the capability to produce simple parts at a much smaller scale than 100nm.

@Polynomial This x-nm technology means the size of a single transistor, a simple switch which is still far from a minimal functional element.

I'm aware of this, but transistors are highly complex in comparison with a mechanical device. In the case of producing very fine filters and sponge-like structures, 20nm is no big deal.