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What is the role of tracrRNA in CRISPR-cas9?
From what I understand, in a CRISPR cas9 complex, gRNA is comprised of tracrRNA and crRNA.  I've read that crRNA is the part which is matched to the DNA which is targeted, but what role does tracrRNA play in the process?  That I'm not clear about.  Also, unless this should be its own question; how is gRNA prepared with cas9 protein?  Does the protein automatically bind to certain RNA strands?

Thank you for your answer.  I have read some of the articles you've given (the link for the second one didn't work).  I've also looked up some other resources; from what I understand, the tracrrna is like an RNA scaffold of sorts, which keeps the complex in active form and which recruits the crRNA to the cas9 molecule.  Based on the first paper, am I to conclude that cas9 proteins isolated from certain species each have certain tracrrna sequences that could be sued with any crRNA (like cas9 proteins from different species recognizing certain PAM sequences)?

Sorry, I have fixed the second link. You are on the right track, the tracrRNA does act like a scaffold. You are also correct that Cas9 homologs from different species appear to have tracrRNA sequences quite divergent from one another. However they must have sequence complementary to the pre-crRNA in order to form the complex so can't be used with any crRNA.

Also, the PAM site recognition does appear to be dependent on the Cas9 in use (If you are interested check out 10.1038/nature13579 and 10.1038/nature14592). This places additional constraints on the pairing of tracrRNA and crRNA.

Your two questions are related and you are correct in your supposition that the Cas9 protein associates to a specific RNA sequence. That of the tracrRNA processed with the crRNA into a gRNA. This is achieved without any other aid so could be considered to  do so automatically. How specific the tracrRNA sequence needs to be is an interesting and often overlooked question.
When comparing tracrRNA from different species it is clear that there is not a lot of sequence conservation but they are predicted to be contain similar secondary structures.
Matching tracrRNA and Cas9 from closely related species does result in in vitro DNA cleavage but this does not work with more distantly related species. Nevertheless they are remarkably versatile and this has been exploited in a number of exciting genome engineering applications where additional structures are added without compromising the binding of the RNA to the Cas9.
The tracrRNA and the crRNA together form the gRNA which targets the Cas9 protein to the location of cleavage. In the endogenous bacterial systems the gRNA takes the form of a processed RNA duplex but in the genome editing implementations this takes the form of a single chimeric RNA with the two components already fused together.
When it comes to actual cleavage of DNA it is important that the Cas9 is in the correct conformation and changes in this are known to occur when binding to the target DNA. Recent work suggests the tracrRNA plays a role in maintaining the Cas9 protein in an active form allowing it to be able to target DNA.
It likely also contributes to the overall stability (and thus efficiency) of the CRISPR-Cas9 complex.
Ultimately the tracrRNA is an essential component of the CRISPR-Cas9 system via it's role in both guiding the Cas9 protein to its target and aiding it's function.
The following references might be of interest to you:
The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems
Guide RNA engineering for versatile Cas9 functionality
Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease

In short, tracrRNA keeps the CRISPR-Cas9 complex (of RNA and the Cas9 protein) catalytically active:

Image source: Figure 5 from Lim, Y. et al., 2016. Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease. Nature Communications, 7, p.13350. Available at: http://dx.doi.org/10.1038/ncomms13350.

This was shown by pre-incubating different sets of components together for 20 minutes, then adding the missing component(s) and observing the resulting amount of DNA cleavage activity. For example, in the "pink" experiment, only the Cas9 protein and tracrRNA are pre-incubated together and the crRNA is added just before the 5 minute cleavage time:

Image source: Figure 1 from Lim, Y. et al., 2016. Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease. Nature Communications, 7, p.13350. Available at: http://dx.doi.org/10.1038/ncomms13350.

The green bar represents the Cas9 protein pre-incubated for 20 minutes with both the tracrRNA and crRNA. The pink bar shows that pre-incubating with tracrRNA but without crRNA still results in the same level of activity (measured as fraction of cleaved product). However, when the tracrRNA is not included in the pre-incubation, as in the yellow and blue experiments, the activity is much reduced.


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Is the glomerular filtration rate, per kidney or for both?
We say in a healthy human being the average glomerular filtration rate is about 120 mL/min. Is that for both kidneys together (60+60), or just one kidney?
Pardon me if this makes me sound like an idiot, because I have a feeling I'm thinking about this wrong.

The definition of GFR given in Wikipedia -

Glomerular filtration rate (GFR) is the volume of fluid filtered from the renal (kidney) glomerular capillaries into the Bowman's capsule per unit time.

Since it is difficult to measure the volume entering into each Bowman's capsule, as a proxy, we measure the clearance of a solute that we know isn't going to be absorbed or secreted. For that particular solute,
GFR = (Urine concentration x Urine flow)/ Plasma concentration
The GFR you obtain here is the sum total of volume of all the fluid filtered from all the renal (kidney) glomerular capillaries into all of the Bowman's capsule per unit time.
Obviously, a single Bowman's capsule can't receive 120ml every minute, it is microscopic!!
So yes, the GFR or eGFR we use in clinical practice is the sum total of the function of both the kidneys. In fact, before transplantation, although the left kidney is preferred from the donor (since it is easier for surgery having a longer renal vein), it is more ethical to estimate individual GFR for each kidney and preserving the better functioning kidney. And yes, the total GFR drops after donating a kidney, but the body has sufficient reserve to manage.
If you still have a tough time figuring that out, think of them as many separate taps opened to fill a bucket. One tap for each glomerulus. The net rate of flow is the sum of flow rate in all the taps. The kidney has million such taps! And the taps are being clogged one at a time in chronic kidney disease - decreasing the GFR. And in a transplant, half the taps are suddenly shut down in the donor! But since this is a healthy kidney, the donor adapts and slowly reaches ~70% of original GFR.

Exactly what I was looking for. Thank you, that was very succinctly put!

how is each kidney's individual GFR estimated in a kidney donor?

if a certain molecule is already in the bloodstream (maybe creatinine) doesn't that mean it's already been absorbed?

It's a rate, not an absolute amount, so you wouldn't divide it up like that. It's like if a car is traveling 60 mph, each tire is going 60 mph, not 15.
You'd divide it up to describe the absolute amount, so if the kidneys filter 120 mL, then you could say each kidney filtered 60 mL (although even then each kidney isn't going to function identically).

Thank you, I understand how it's a rate and used as an indicator of the function of the excretory system.
My point is, if you removed one kidney, gradually the remaining one may grow to work more. But theoretically, the day that you remove the first kidney, the GFR is halved?

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Why are there no Nissl bodies in the axon?
What could be the significance of not having Nissl bodies (endoplasmic reticulum) in the axon hillock (a part of the cyton) and axon? Does it have something to do with the processing of action potentials?
What prevents Nissl bodies from entering the axon hillock even though they can enter dendrites (which are much narrower)?

Can you add more details? Otherwise it looks-like homework question.

Looks much better. Nominated for reopening.

Molecularly, Nissl bodies are the densely clustered ribosomes on the ER. The basophilia is due to the RNA in the ribosomes. The question you have raised is a very significant one. For perspective, I quote a book Fundamental Neuroscience, 2002 (2002 is pretty old for neuroscience!)

The hillock is a region where materials either are committed to the axon (cytoskeletal elements, synaptic vesicle precursors, mitochondria, etc.) or are excluded from the axon (RER and free polysomes, dendritic microtubule-associated proteins). The molecular basis for this sorting is not understood. Cytoplasm in the axon hillock does not appear to contain a physical "sizing" barrier (like a filter) because large organelles such as mitochondria readily enter the axon, whereas only a small number of essentially excluded structures such as polysomes are occasionally seen only in the initial segment of the axon and not in the axon proper.

But a recent paper GG Farias et al. 2015, proposed the presence of a special area in the axon hillock known as the Pre Axonal Exclusion Zone (PAEZ) that tries to explain this polarized sorting by differential binding of microtubule motors.

Although the paper mainly talks of the sorting of axonal and somatodendritic vesicles as seen in the picture, they also seem to apply for the RER which actually are the basis for Nissl's granules.

This structure excludes not only somatodendritic vesicles but also larger organelles, such as the Golgi complex and the rough ER, in effect constituting the cytoplasmic boundary for the somatodendritic and axonal domains..... The exclusion of the rough ER and Golgi complex, in addition to somatodendritic vesicles, at the PAEZ suggests that a common restriction mechanism may operate for all of these organelles.

Well, as you might have understood by now, it's not a matter of the size of the axon/ dendrite since same sized vesicles are being diverted in either direction and as previously mentioned, even mitochondria enter the axon.

Logically I would say that since there is more entry and exit of substances at axon hillock , a large organelle like rer would hinder the transportation by its presence at there, also er too needs to be in proximity with the nucleus for its activity , therefor it cannot enter axon and far away into dendrites.

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How specific are CRISPR-cas9 cuts?
CRISPR-cas9 uses a string of RNA that matches with DNA and makes a double stranded cut at that point.
If the RNA is just a few letters in length, the enzyme would cut DNA in many places. It would be unspecific. But if you can make the RNA string very long, it would only cut at the exact place where you want to cut.
So how long can you make the RNA sequences?
And is this enough to avoid unintended cuts?

The gRNA can be anywhere from ~17 to ~27 bases (not including the PAM site), with the average being about 20-23. See here for more info.

The problem of off-targets in CRISPR/Cas is often discussed. It was shown that the system allows mismatches up to five basepairs. For your question, if it is helpful to elongate the gRNA: it was shown that truncating the RNA enhances the specificity more than elongating (see also here).
So what can we do? Well, there are different methods to improve the specificity. The simplest may be the usage of lower dosages of the CRISPR/Cas system. Another method is to use altered PAM motifs.
A more complicated method is to convert the Cas9 into a nickase. In this strategy you would use two different gRNAs for two different target sites and introduce ssbreaks. A modification of this method is to completely knock-out the enzymatic activity of Cas9 and fuse it to a FokI nuclease which will cut the DNA only when it dimerizes.
There are several advantages in these methods, however, you may also loose on-target effects.
There is another method which, in my opinion, is really awesome. It is possible to alter the energetics of the DNA binding site resulting in high-fidelity variants of Cas9 (HF1 and HF2)
So, I hope I could help you. There are other and even more effective ways to increase CRISPR/Cas specificity instead of altering the gRNA. If you're interested, have a look on the references.

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CRISPR-Cas Systems
In the context of the bacterial systems (not the gene editing tool), I was wondering what happens to the foreign DNA after the Cas proteins have created a new spacer.
It is really not clear to me, most of the documentation I have found focuses on the subsequent steps (expression and interference) and do not discuss the fate of said foreign DNA. For example, see this image from wikipedia https://en.wikipedia.org/wiki/CRISPR#/media/File:Crispr.png

It's not really clear to me what you mean.  Do you mean what happens to the foreign DNA or what happens to the spacer?  The former is cleaved to form a spacer, the latter is inserted into the genome.

Ok thanks, how do you know that it is cleaved? Are there endonuclease activities in the proteins that deal with the foreign DNA? I was under the impression that the spacer was created by copy of the protospacer of interest.

@AlterSid Cas is that endonuclease. Please read about CRISPR-Cas and ask specific questions if you have any. Wikipedia is good enough.

Try this paper http://www.ncbi.nlm.nih.gov/pubmed/20056882 it makes a nice overview of the all process and if you want to go deeper check the references, they cover all the passages involved in the CRISPR mediated immunity.

Do CRISPR systems attack the entire DNA of the virus or certain specific DNAs (dictated by the spacers)?

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CRISPR Knock in
Using the CRISPR/Cas9 technology, it is possible that after inducing a DSB with the Cas9 endonuclease guided with an RNA designed by the user and using a template DNA, get a desired Knock-In (KI) by homologous recombination.
I have read that for desired KI-s that are less than 200 bp, a olygonucleatide should be enough as template. On the contrary, if the desired KI is bigger than that, a plasmid vector should be used.
Is that information accurate and correct? and, where is the upper limit for the KI length?

Successful knock-in of 1000 bp and 500 bp has been done before. ncbi.nlm.nih.gov/pmc/articles/PMC5307959/


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Mutations/deletions with CRISPR
I need to stop some protein from being active and searching for some universal way to do so. In mammalians.
With CRISPR it is possible to knock-out the entire gene. But it's a little complicate (need two gRNA for eg.). There are also another reasons, so I need to do this with only one gRNA.
So I am thinking about inserting some mutation.
My question: Is there way to make some mutation that will prevent creation of functional protein, with high efficiently - with one gRNA. And without need to know the active site of protein.
For example, frame-shift or mutation around start codon? Is it possible? If so, how can I do this? To where I need to send the gRNA?
Thanks.

First of all, If you talk about system, please specify if it is cell line or animal. Secondly, if you say "There are also other reasons", specify them too. Also, what do you mean by "without need to know the active site of protein"?

Well, synthetic CRISPR/Cas systems do have an option of having a single guide RNA (sgRNA)

You can do with a single gRNA. All that CRISPR-Cas, ZFN or TALEN systems do is to introduce a double strand break at a specific site. The DNA gets repaired via two mechanisms — non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ is error prone and it may introduce indels that can compromise with gene function (frameshifts etc). While HR can be used to integrate a transgene or repair a mutation, NHEJ can be used to create mutations.
I haven't really knocked out a gene using CRISPR-Cas but it is good to target the initial part of the ORF. There will be many other factors too that will determine what site should be chosen for targeting and an important factor would be the uniqueness of the target sequence. See this site for guidelines on selecting a good target sequence.
You would have to do rigorous screening nonetheless; the advantage of insertional knock out is that screening can much be easier (for e.g. by inserting GFP or puromycin-resistance gene).
You can have a look at this paper and the supplemental information; they have targeted 18080 genes with a CRISPR-Cas library.

Huge disadvantage  of insertion is very poor successful rate. Less then 5%. So I still looking for the answer. Where in the gene to cut, to make protein not working. About ORF, I wrote about start codon, but need more information and sources. Where exactly. Thanks.

And about the uniqueness, off topic question, is it possible to make a gRNA that will target a lot of different places in genome? Didn't heard about that. Nonspecific gRNA?

@VGranin the insertion rate you refer to is inserting a specific sequence, a non-specific indel mutation to cause a frameshift like WYSIWYG is talking about can have success rates upwards of 70%. And the only way you could target a lot of different sites with a single gRNA is if all the sites had a sequence in common, gRNAs are specific to a given sequence.

@VGranin As I said you can use a single gRNA to make the cut. Definitely ahead of the start codon but not too far off (if frameshift on non-sense mutation happens then you  will just have a little bit of the original sequence). So you have to optimize between closeness from start codon and other factors such as unique site and others mentioned in the link that I have given in the answer.

@Luigi. Thank you. But he mention a GFP insertion... Anyway, what CAS9 do is just making break. A non-specific insertion it is not something that we do, it's the cell way to repair. What I need, is a place to target gRNA to make a break that will make protein non working.

@Luigi Are you sure about the 70%; it depends a lot of factors including cell type and cell cycle stage amongst others.

@VGranin I mentioned that you can do an insertional mutagenesis too which, although will require two gRNAs, make the screening much easier (you can FACS sort the knockouts).

@WYSIWYG for a straight ds knockout, we usually see between 40% and 70% successful depending on the target

@Luigi perhaps we should discuss this on chat but which cells are these?? And how do you screen? PCR? Screening is the most irritating thing

@ WYSIWYG, thank you. So near the  start codon. But is there some paper that did this in different situations and proteins - and show some numbers of success e.t.c? Cause I can't find.. Thanks.

We use puromycin resistance to positively select for transfected cells, after this selection (with transfection efficiency now 100%) is when we see 40%-70% success with PCR screening (which is definitely a pain). @VGranin this paper will tell you everything you ever need to know about performing CRISPR knockouts

@VGranin this paper too. They have done a genome scale knockout for 18080 genes (not at the same time of course)


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Splice in with CRISPR/Cas
I need to splice a gene into a human cell genome, with highest rate possible. I mean, doesn't really matter where the gene enters, nor does it matter if some cells die as a result of this.
CRISPR know to knock-in genes with very high specifically, this reduce the success rate if we have a low amount of gRNA and/or of the protein.
I need to insert the gene, without the need of targeting some specific place.
Is this possible in some way with CRISPR?
I know that there may be better technique to do this, but I can only use CRISPR.

Is there a good way to detect the gene product in a living cell? Because you could just sort the cells by FACS and take the cells that have the knock in, then grow those.

Could you please define what the question actually is? If it is "can you use CRIPR/Cas9 to insert a gene in a genome?" Yes you can.

@cagliari2005,  The question is, is it possible to insert a gene with Crispr with high success rate,  when I have a very low amounts of the Crispr in the cell? I mean I actually need to reduce his high specifically - to get higher rates. Is it possible ?

What determines the specificity and cleavage efficiency is the guide RNA. As you cannot act on that I personally don't know any tricks on something like cell culture conditions which would do what you want.

Maybe you could look at conditions that increase homologous recombination rate.

Using a chemically modified guide RNA with higher melting point might help with the insertion rate, but I suspect it would also decrease the specificity.

Decrease the specificity - it's what I need, cause it will higher the insertion rate. But may you tell more about the melting point of gRNA?..

I'm not an expert at RNA backbone modifications, but I have read that Locked Nucleic Acids or ZEN modified oligos can increase the melting point. IDT may be helpful for designing the oligo, but be prepared to spend a lot on extensively modified RNA oligos.

A paper was published about a week ago in Nature Biotechnology and adresses your question, Maruyama T et al., 2015. I must say I found the authors' strategy extremely clever.
It is not about increasing efficiency by reducing specificity, but simply increasing efficiency (which is your ultimate goal anyway). What the authors did was to inhibit nonhomologous end joining (NHEJ) to promote homology-directed repair (HDR), two DNA repair mechanisms that compete in cells and of course HDR is the mechanism needed for the CRISPR/Cas9 system.
They achieve NHEJ inhibition using the molecule Scr7, a DNA ligase IV inhibitor which in turns perturbes NHEJ.
Using Scr7 they boosted by 3 to 19 fold (depending on the cell line) the insertion of the target gene. Here the graph showing these results
See paper figures
Moreover using 1μM of Scr7 over 24h on DC2.4 cells increased the % of transfected cells from 4.58% to 58.3%, a neat ~13-fold increase. Here their results:
See paper figures
Hopefully this should give you some ideas.

Interesting answer.

Looks like they also used single stranded DNA oligos for some of their short insertions (see supplementary table 7), consistent with my speculation based answer below. The longer inserts are still double stranded. Would be interesting to see if long single stranded DNA would insert more efficiently too. Just glad to see I was speculating in the right direction.

@cagliari2005, wow, thank you!! Really great.

Just a side-question: is it fine from a copyright perspective to publicly post diagrams from articles?

@TMOTTM It is not plagiarism as I am citing the source but regarding copyright infringements I think you are right in pointing that out. As those figures comes from Nature Biotechnology I am not supposed to publicly post them (I actually removed them) - see Nature policy. If they were coming from open-source journals (say PLoS one) then you can as they are publicly available.

I don't know, I was just wondering.. anyway, i can still see the figures, just in case.

@TMOTTM Now you can't ;)

I'm going to preface this answer with the disclaimer that I have never used CRISPR/Cas, and there is a fair amount of speculation here.
But I think that efficient CRISPR mediated knock-in probably has three parts, targeting, integration, and selection.
The targeting is accomplished by the guide RNA, which is often introduced as DNA on the same plasmid that codes for the Cas9, or can be in vitro transcribed or synthesized as delivered as RNA. However, a poster suggests that a properly designed DNA fragment can be delivered and produce sufficient amounts of guide RNA in situ to produce efficient cutting.
The integration step is where the template DNA is inserted into the double strand break by homologous repair. Homologous repair efficiencies vary greatly from cell to cell, and is often less efficient than non-homologous end joining. However, using a single-stranded DNA might increase the efficiency.
However, even if only a small percentage of cells is correctly edited, it may be possible to select those cells and grow them. NEB describes a Cas9-GFP fusion (page 4) that can be used to bind GFP to a certain DNA sequence. It may be possible to produce CFP and YFP variants of this construct and use FRET to detect cells with the knock in. (This is where the speculation comes in) With the two fusion proteins and two guide RNAs for adjacent sequences in the knock-in gene, you might be able to detect cells where a CFP and YFP cas9 are close enough that you get FRET. This might allow you to sort cells by FACS, and then grow those cells.
Of course if your knock-in is making a membrane bound protein exposed to the surface, you could just use labeled antibodies to detect that protein.

Thank you, but I don't ask how to separate the cells. I ask how to force Crispr to insert in higher success rate. How to make him less specific or what regions to target. If it's possible. And your second link about Zinc-finger nucleases and not Crispr.

The second link is about homologous recombination. CRISPR and zinc-finger nucleases are just about cutting the DNA, homologous recombination is how the new gene is actually inserted. And I don't think you're going to easily push the CRISPR success rate above 50% of all cells, the recombination rate will probably be lower. A good selection strategy can find those recombined cells and give you a population with 100% recombination.

If you want to have random integration you need to find gRNA sequences that are nonspecific ; everything built for CRISPR so far has been geared towards precise integration, not random integration.
Talk to an informatician about pulling out non-unique sequences from the human genome. You will then need to clone out a library of 20nt sequences that end with a PAM sequence (NGG) and chuck them into cells with another construct encoding a Cas9 and the insert.
Of course, if you simply need integration you don't need CRISPR either - you can use any bog standard lentiviral vector and you will get a stable cell line made with the gene inserted in random places. Using CRISPR for this overcomplicates things greatly.

OP says he "can only use CRISPR".

Yes I am well aware; I am also aware that this is a massively complicated and probably a very ineffective way of doing things. Not least because spiking something in with a CRISPR targeting strategy often requires homology arms of up to a kb; and in this scenario, it means one has to not only make a library of sgRNAs but also a library of his insert flanked by 1kb sequences for every locus he is trying to target.


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Why don't bacterial cell walls prevent bursting when attacked by the complement?
The complement system creates pores in cell membrane which leads to influx of lots of water thereby causing lysis of bacterial cell.
But what I fail to understand is that if bacteria have cell walls around them, why will they burst ? Shouldn't they simply become turgid just like plant cells do ?

Cell wall is porous enough to allow water and other small molecules to pass. Influx of water will still destabilize the cellular environment and in essence rupture of the cell. Bursting, IMO is quite a dramatic term for this.

Although this seems to be a simple question on the exterior, the answer is going to be confusing and unclear because we still don't know the answer exactly.
Short answer - MACs are bactericidal not because of water gushing in. It is due to other mechanisms effected by damage to membrane integrity.
Long answer
First of all, I am assuming you already know the complement cascade, the pathways and the like, and I won't get into it. But to mention in passing, MAC is not the only way the complement can help the immune system. It can also opsonise antigens and release anaphylotoxins to modify the immune response.
Most studies concerning the assembly of the MAC and the resulting membrane lesions have been conducted in model systems, e.g. erythrocytes and artificial lipid bilayers. The advantage of these systems is the ease of characterizing the membrane lesions compared to bacterial membranes. Also, complement fixation tests (which use erythrocytes and complement) have been widely used even diagnostically until not very long ago. The osmotic damage, ion effluxes that you talk of happen here and leads to the lysis of erythrocytes. Even in their case, there are 2 models of how MACs work - one is by making a pore (in the pic) and the other is by forming leaky patches in the membrane. However, the way bactericidal MACs work is thought to be different and a little more complex than this -

This picture may have been used for just didactic purposes but cannot be applied in a bacterial system simply because bacterial cell envelopes are different. The two major groups - Gram positive and Gram negative cell walls are important to differentiate while discussing the mechanism of MAC bactericidality. It is important to note that MAC mediated bactericidal action has been reported predominantly only in Gram negative organisms. This resistance in Gram positives is thought to be not because of the osmotic turgor or turgidity but because of the thick peptidoglycan in Gram positive bacteria, preventing access to the inner cell membrane where the MAC assembles. There are MAC resistant Gram negative bacteria too, but let's not discuss that here. It is not off-track to mention here that species like Neisseria where the capsule is thin and the peptidoglycan is too, are more susceptible to complement damage.
So, assuring that the MAC mediated bacterial lysis is not just water gushing in, how is it thought to happen??
For this, we need to revisit the structure of the Gram negative cell wall. The cell wall structure is quite intriguing since there happen to be two lipid membranes enclosing a periplasmic space which contains little peptidoglycan that Gram positives have much more.

Since there are two lipid membranes, technically, there's a possibility that the MAC can be formed on both these layers (MAC's assemble on hydrophobic membranes). MACs assembling on the OML (outer membrane layer) are not sufficient to cause lysis. It is confirmed that the damage has to occur to the IML (inner membrane layer). Inner membrane damage is thought to be bactericidal since it is more restrictive in its permeability than the OML. Also, the IML is linked to several important cellular, metabolic functions that require proper ionic gradients. One example is the oxidative phosphorylation that takes place around the IML.
This should answer your question in that the mechanisms of cell lysis seem to be slightly different in bacteria than the model systems (erythrocytes), but let us delve a little deeper into this.
One persistent question is "how can something as huge as the MAC, penetrate the OML and the peptidoglycan layer, to reach the IML to cause damage?" The hypotheses that MAC just forms a hole in the OML and other serum factors seep in and cause damage to the IML has been ruled out since complement alone can be bactericidal in nature. To explain this, two models have been proposed -
One, where the complement attaches at special sites in the membrane where both OML and IML approximate (at special sites where large proteins span both membranes) and another where, the complement creates a pore to allow C9 monomers to leak in, in turn polymerizing to damage the IML.
I assume this explains it all. Still, I implore you to refer to the link below and correct me if I've been wrong anywhere.

Reference : The bactericidal mechanism of the complement membrane attack complex, Lars Ootes, (PDF)


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How does coconut water solidify into hardened coconut meat?
At first, the coconut is filled with liquid endosperm. Later when it ripens, the outer layers form brown fibrous covering, and the inner solid layer turns brown and hardens. Within that, a white layer forms (which is soft and thin at first and then gradually hardens and thickens). After ripening, there is a white layer with very little fluid. So, it's gotta be the liquid endosperm which has hardened over the course of time to form the white layer. So, how does it harden?

is it just an aqueous solution?  if so, evaporation?

@dbliss How does liquid evaporate in closed environment? And how does the white layer form due to evaporation?

There are three types of endosperms encountered in botany - nuclear, heliobial and cellular. The endosperm of Cocos nucifera is both special and interesting.
Initially, the cocunut is a nuclear liquid endosperm. Meaning, it is a coenocytic liquid tissue with lots of rapidly dividing nuclei. The reference I cite here (although pretty old), states that it is safe to assume the fluid to be the 'sol' phase of cytoplasm. At some point, gelification of the sol surrounding the nuclei occurs and cellular boundaries start to appear.
These cells and nuclei start to settle down on the endothelium (inner lining of the integuments) and the cells divide to produce a columnar sort of a covering over the it. This is what forms the characteristic coconut meat. Development of the cellular endosperm is by coalescence of these deposited cells and nuclei at the antipodal end (bottom end of the fruit) and continues towards the top end. You might have noticed that the meat is thicker at the bottom but thins out towards the top.
The cells right above the endothelium stay meristematic for a long time while the columnar cells on top of this meristematic layer grow radially inward.

If the terminology seems confusing, the bottom end is the antipodal end. End. in the diagram stands for endosperm - the coconut meat. The Sh. is for the shell, which is the thickened inner integument. The inner lining of this shell has been referred to in this answer as the endothelium. Mic. stands for micropyle where the stalk (pedicle)  of the fruit is. It is at the micropylar end that the embryo is placed. Remember! The coconut meat is not the growing embryo.
How does the meat harden?
As said, initially the deposited mass of cells and nuclei is jelly like and slowly hardens after proper cell wall synthesis etc. The hard white coconut meat that can be grated, is end stage of maturation and is formed when the above said tissue architecture has been established.
What happens to the coconut water?
After the cellular endosperm has reached its final state a little water is still left in the coconut. This water is opalescent compared to the clear water found in younger fruits. Interestingly this water is now totally devoid of nuclei.

Whether the decrease in liquid is the result of its incorporation into the endosperm cells or due to evaporation due to drying and cracking of the pericarp remains uncertain.

Sorry for the anticlimax. But this has been quoted from a 1955 paper. I was unable to find newer papers. Little work has been done in morphological botany in the recent past, but I'm pretty optimistic the answer would lie somewhere in literature.
References:

V.M. Cutter, Jr., K.S. Wilson and B Freeman. Nuclear Behavior and Cell Formation in the Developing Endosperm of Cocos nucifera. American Journal of Botany (1955), 42 (2), 109-15.
A. Abraham, P.M. Mathew. Cytology of Coconut Endosperm. Annals of Botany (1963), 27 (3): 505-512.
The Hindu (2002) : Mystery behind coconut water, R. Subramaniam.

Coenocytic is used when there are multiple nuclei where cytokinesis is not followed by karyokinesis. In a syncytium too, multiple nuclei are found but here, it is formed by fusion of cells leading to loss of their boundaries. This terminology is used quite loosely in literature.


text:
Size of classical liver lobule across species
A classical liver lobule is made up of a central vein and the portal triads.  A typical human liver weighs around 3 lbs. , while a typical bovine liver weighs around 12 lbs.  I was wondering if anyone had a reference that might discuss the size of a bovine liver lobule vs. the size of a human liver lobule.  Specifically, is the bovine liver lobule also larger than a human liver lobule?


text:
Why is Leptospirosis so difficult to catch?
I was on a small mammals training course and the professor was warning of the dangers of Leptospirosis. Apparently 5-40% of Rats have it and an unknown proportion of other animals. The warning sheet they handed out listed farmers, gardeners, etc as 'at risk' groups. I looked it up on the NHS website out of interest but it said there were only about 25 cases a year in the UK, even of the mild form.
I understand that it dies when it's dried out, but still, in notoriously rainy Britain, I can't seem to make sense of the numbers. There are at least half a million farmers in our country, probably a similar amount of gardeners, at least several thousand homeless and poverty stricken who must live in very close proximity to the huge urban Rat population, not to mention the potential for rodents to get into domestic water tanks. In rainy weather everything can stay wet for days. Also, if I remember rightly, an Olympic athlete recently died from it, so it obviously doesn't require a weak physiology to get in.
So, the question is, given what seems to me to be a massive prevalence (40% of all Rats), vast contact opportunities (all land workers during spells of wet weather), and seemingly a tough little organism (can kill an Olympic athlete), why is it so rare (25 cases in 70,000,000 people)?

Leptospira, the bacteria is largely found in the urine. So the urine from the rat has to get into cuts or wounds of a human. That seems rather unlikely. For such a thing to happen, you need ample rains, improper sewage and lots of rats. So apart from occupational groups, the disease also has an epidemic nature. During heavy rains in Mumbai, rodent infested sewers overflow and there's sudden spike in leptospirosis cases. I don't know the nos. but my micro txt calls it the most widespread zoonosis. Spells of wet weather aren't enough, there needs to be water logging combined with contamination.

Yes, Leptospirosis is, of course, a major problem in tropical areas, but as far as I know, there has never been an epidemic in Britain, yet we have some floods as well as all the other things I listed in my question. What puzzles me is there seems to be nothing in the bacterium's characteristics or mode of transmission that would account for the lack of epidemic in Britain. A little further reading I've done since asking the question seems to even imply that it can survive in moist soil http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/msds95e-eng.php, we shouldn't even need a rainy season.

An epidemiologist must be able to answer this question but id like to point out that the presence of bacteria doesn't necessarily lead to infection. Something as ubiquitous as Clostridium tetani in the soil doesn't cause tetanus in everyone (even in pre-vaccination era).

That's true, but the mode of transmission with C.tetani is what prevents it from reaching epidemic scales, it requires a relatively deep wound. What I seem to be missing is the equivalent characteristic of Leptospires which prevents Leptospirosis from reaching epidemic proportions in Britain.

Both deep and superficial wounds can cause tetanus. In recent times superficial wounds have become the main mode infection, more than deep wounds. This may be because deeper wounds receive more attention. I know this is off-track but still...

No, not at all off-track, I see what you're saying, it may indicate that other pathogens share whatever it is that Leptospires have which make them more difficult to catch than their prevalence in the environment would at first suggest. Do you happen to know if Tetanus has a mild form? One theory I considered with Leptospirosis, is because it has a mild form, it is under-recorded and in fact thousands of people are catching it without even noticing (this would then tally with the prevalence). Of course this just moves the question to why the severe form is so rare.

Any inputs? @theforestecologist

Based on some climate informatics, however, using our examples of Mumbai and I'm going to use London here: It's always warmer in Mumbai, closer to an average of 30C compared to ~20C in London. And when it rains in Mumbai, it rains: 60-70mm rain average in London compared to 600mm or greater during the months it actually does rain in Mumbai. I'd also consider healthcare standard. There are many things in play that need considering.

Yes, I've read that the disease is more of a problem in Tropical areas, but I've not read anything to indicate why. As I said in my post, it is not killed by cold weather (certainly not 10-20C anyway) and it can already survive for many weeks in just damp mud, it doesn't need any rain at all let alone 600mm. What I'm really after is why, from a physiological point of view, the bacterium is so much worse off in temperate zones.

Let me simplify the question. Rodents getting into the cold water tank is a problem described by various plumbing books as "quite common". People, wash, bathe, brush teeth and occasionally drink this water. The incidence of Leptospirosis from unknown sources in the UK is fewer than ten (eliminating foreign travel, water-sports and occupational exposure). So the question is, how do we get from a "quite common" situation which provides direct water-bourne exposure to rodents, in a country of 70,000,000, to fewer than ten (if any) cases of Leptospirosis caused by this type of exposure?

I see no-one has been able to answer this, but just in case anyone is considering the question, I've just found an interesting set of data from the national archives on reported zoonosis in the UK. It records the ages, and often causes, of all reported cases of Leptospirosis. Using age as a proxy for domestic sources (a domestic source would affect all ages equally) it would seem, as far as records go back, that no-one has ever contracted Leptospirosis this way, so I'm looking to explain not just a low chance, but no chance at all.


text:
Similar smell for avocado and raspberries
I noticed when heating frozen avocado that the smell was just like fresh raspberries in the summer. Does anyone know what makes them smell so similar ?
(https://doi.org/10.1016/B978-0-12-408117-8.00029-5)
"One-half an avocado is a nutrient and phytochemical dense food consisting of the following: dietary fiber (4.6 g), total sugar (0.2 g), potassium (345 mg), sodium (5.5 mg), magnesium (19.5 mg), vitamin A (5.0 μg RAE), vitamin C (6.0 mg), vitamin E (1.3 mg), vitamin K1 (14 μg), folate (60 mg), vitamin B-6 (0.2 mg), niacin (1.3 mg), pantothenic acid (1.0 mg), riboflavin (0.1 mg), choline (10 mg), lutein/zeaxanthin (185 μg), cryptoxanthin (18.5 μg), phytosterols (57 mg), and high-monounsaturated fatty acids (6.7 g) and 114 kcals or 1.7 kcal/g (after adjusting for insoluble dietary fiber)"  ("Hass Avocado Composition and Potential Health Effects", Mark L. Dreher and Adrienne J. Davenport)
https://www.sciencedirect.com/topics/food-science/avocado
$
\begin{array}{|l|c|c|}
\hline
  \text{Compound} & \text{Raspberry} & \text{Avocado} \\
\hline
   \text{provitamin A/$\beta$-carotene} & & \checkmark \\
\hline
   \text{vitamin A} & \checkmark & \checkmark \\
\hline
   \text{vitamin B} & \checkmark &  \\
\hline
   \text{vitamin $B_1$/thiamine-HCl} & \checkmark & \checkmark \\
\hline
   \text{vitamin $B_2$/vitamin $G$/riboflavin} & \checkmark & \checkmark \\
\hline
   \text{vitamin $B_3$/vitamin $PP$/niacin} & \checkmark & \checkmark \\
\hline
   \text{vitamin $B_5$/pantothenic acid} &  & \checkmark \\
\hline
   \text{vitamin $B_6$/pyridoxine} & & \checkmark \\
\hline
   \text{vitamin C/ascorbic acid} & \checkmark & \checkmark \\
\hline
   \text{vitamin D/calciferols} &  & \checkmark \\
\hline
   \text{vitamin E/$\alpha$-tocopherol} & \checkmark & \checkmark \\
\hline
   \text{vitamin $H$/biotin}  &  & \checkmark \\
\hline
   \text{vitamin K_1/phytyl menaquinone} &  & \checkmark \\
\hline
   \text{acid:citric acid} & & \checkmark  \\
\hline
   \text{acid:folic acid} & \checkmark &  \\
\hline
   \text{acid:malic acid} & & \checkmark  \\
\hline
   \text{acid:oxalic acid} & & \checkmark  \\
\hline
   \text{$\alpha$-carotene} & & \checkmark \\
\hline
   \text{anthocyanins} & \checkmark &  \\
\hline
   \text{choline} &  & \checkmark \\
\hline
   \text{cyanidin-3-glucoside} & \checkmark & \\
\hline
   \text{anthocyanin was cyanidin-3-rutinoside} & \checkmark & \\
\hline
   \text{cryptoxanthin} &  & \checkmark \\
\hline
   \text{dietary fiber} &  & \checkmark \\
\hline
   \text{ellagitannins} & \checkmark &  \\
\hline
   \text{folacin} &  & \checkmark \\
\hline
   \text{folate} &  & \checkmark \\
\hline
   \text{high-monounsaturated fatty acid} & & \checkmark\\
\hline
   \text{phospholipids} & & \checkmark\\
\hline
   \text{glycolipids} & & \checkmark\\
\hline
   \text{free fatty acids} & & \checkmark\\
\hline
   \text{lutein/zeaxanthin} & & \checkmark  \\
\hline
   \text{persenone A} & & \checkmark \\
\hline
   \text{persenone B} & & \checkmark \\
\hline
   \text{phytosterols} & & \checkmark \\
\hline
   \text{$\beta$-sitosterol} & & \checkmark \\
\hline
   \text{sugar} &  & \checkmark \\
\hline
   \text{B (boron)} &  & \checkmark \\
\hline
   \text{Ca (calcium)} &  & \checkmark \\
\hline
   \text{Fe (iron)} & \checkmark & \checkmark \\
\hline
   \text{K (potassium)} & \checkmark & \checkmark \\
\hline
  \text{Mg (magnesium)} & & \checkmark \\
\hline
   \text{Na (sodium)} &  & \checkmark \\
\hline
   \text{P (phosphorus)} &  & \checkmark \\
\hline
   \text{Zn (zinc)} &  & \checkmark \\
\hline
\text{2(R)-(12Z, 15Z)-2-hydroxy-}\\\text{4-oxoheneicosa-12,15-dien-1-yl acetate} & &\checkmark\\
\hline
\end{array}
$
$
\begin{array}{|l|c|c|}
\hline
   \text{sugar} & \text{Raspberry} & \text{Avocado} \\
\hline
   \text{glucose} & & \checkmark\\
\hline
   \text{fructose} & & \checkmark\\
\hline
   \text{d-Manno-heptulose} & & \checkmark\\
\hline
   \text{perseitol} & & \checkmark\\
\hline
   \text{sucrose} & & \checkmark\\
\hline
\end{array}
$
$
\begin{array}{|l|c|c|}
\hline
   \text{neutral lipid} & \text{Raspberry} & \text{Avocado} \\
\hline
   \text{monoacylglycerols} & & \checkmark\\
\hline
   \text{diacylglycerols} & & \checkmark\\
\hline
   \text{triacylglycerols} & & \checkmark\\
\hline
\end{array}
$
$
\begin{array}{|l|c|c|}
\hline
   \text{neutral lipid:triacylglycerol} & \text{Raspberry} & \text{Avocado} \\
\hline
   \text{dioleyl palmitin} & & \checkmark\\
\hline
   \text{triolein} & & \checkmark\\
\hline
   \text{dioleypalmitolein} & & \checkmark\\
\hline
   \text{linoleyl oleyl palmitin} & & \checkmark\\
\hline
   \text{linoleyl diolein} & & \checkmark\\
\hline
\end{array}
$
Seems there are hundreds of volatile compounds for raspberries, so will not list them all here (unless I find a matching volatile compound, then I might update this question).
What I found matching http://dx.doi.org/10.1590/1678-457X.01316 and doi:10.3390/molecules20022445:
(E)-3-Hexen-1-ol    Green, grass, leafy odor; bitter, grassy-fatty notes
1-octanol   Grassy, plastic, solvent
2-butanol   Oily, wine-like, fusel-alcoholic note
2-ethyl furan   Strong, sweet-ethereal, burnt brown musty odor
2-decanone  Orange-like, floral odor
2-hexanone  Fruity, ketonic, banana, yeasty, cheese, dairy notes
2-phenyl ethanol    Rose, perfumery
3-methyl-3-buten-1-ol   Fruity, green
4-methyl-1-pentanol Oily green-fruity, herbaceous,
acetophenone    Sweet, pungent, harsh
benzaldehyde    Almond, fruity, powdery, nutty
benzyl acetate  Sweet, floral, fruity odor of jasmin and gardenia, fruity taste
benzyl alcohol  Faint, sweet, fruity aroma; sweet taste
ethyl acetate   Ethereal, sharp, wine-brandy like odor
hexanal Oily, avocado
limonene    herbal, sweet
linalool    Sweet floral
naphthalene Musty taste
nonanal Fatty-floral-rose, waxy odor
octanal Fatty-fruity odor; sweet, citrusorange-fatty taste
p-cymene    Strong, characteristic, terpene odor
pentanal    Strong, acrid, pungent odor; chocolate
α-copaene   Woody, spicy, sweet
α−terpineol Sweet, floral (lilac), lime odor;
β-pinene    Sweet, lemon
γ-nonalactone   Strong, fatty, coconut

Attempt enhancing the quality of your question and, firstly of your research

@Algae — The question is clear (despite being flagged as otherwise) and the poster has done some research which he believes will help someone provide an answer. I personally regard his question as uninteresting, and regard him as naive in thinking that any common nutrient in his lists is likely to be responsible for his (subjective) observation. He would be better advised to search specifically for the biology of odorants, but I don't think your comment is particularly fair or helpful.

Since listing nutrients was discouraged I will stop now ...

@Emil Although I do not know the answer to your question, I disagree with David & think that this is exactly how biochemists identify tastes & smells. Impossible food wanted a meat-free burger so they put beef into a chromatography-mass spectrometry machine, which identifies specific molecules. Among other things, they detected butter, syrup, and used diapers. Before advanced mass-spec, quantifying the smell was a challenge that has been going on since the 1800s. Here is a paper from 1963 suggesting one method to quantify the smell by using many people.

I have ordered a book called Smellosophy now, hopefully I will get a better understanding of the psychology after reading that one.


text:
Understanding the brain: how are neurotransmitters released in the brain?
I have a basic knowledge of how neural networks work. A potential difference is created that forces sodium, potassium, chloride, and calcium ions to flow which carries an electrical signal to the end of a synapse. From there, the presynaptic neuron releases neurotransmitters creating a potential difference to the postsynaptic neuron.
What I don't understand yet are the mechanics of which neurotransmitter will be released. Do the vesicles of each type of neurotransmitter have an energy threshold? If yes, when the highest energy threshold of a vesicle is achieved, does this mean all the other vesicles will release the encapsulated neurotransmitters?

The vesicles are associated with a calcium-sensitive membrane complex composed of quite a few proteins, some details here. (I'll try to detail an answer when time permits)

Have you heard of SNARE proteins?

The release of neurotransmitters is a very specific type of SNARE mediated exocytosis. The action potential from the influx of sodium propagates along the axon and reaches the axon terminal, containing the calcium channels. This causes the calcium cations (ions) to travel down the electrochemical gradient. The Ca2+ then binds to a protein. The vesicles containing the neurotransmitter called synaptotagmin (Ca sensors), would cause conformational changes leading to the formation of a SNARE complex. Some good animations can be seen here and here.
Regarding whether there is an energy threshold: there is a threshold on the amount of Calcium needed to cause the activation of synaptotagmin, as with all types of SNAREs.
See this article: http://www.pnas.org/content/93/23/13327.full.pdf
Past a certain calcium threshold, all the neurotransmitter should in theory be released, but whether they cause a response would depend on the receptor and on the postsynaptic membrane, as each synapse is specific in the neurotransmitters and receptors it expresses.

The mechanism was nicely described by FZG, I'd just like to add:

the mechanics of which neurotransmitter will be released. Do the vesicles of each type of neurotransmitter have an energy threshold?

According to this book, one such mechanism is frequency of stimulation (low frequency of action potentials) and subsequent Ca2+ distribution. With low frequency of stimulation, channels closer to the cleft are open, which means, there is higher concentration in the proximity of the cleft. With higher frequency, the concentration in the terminal bud is more equal in the proximity and further away from the synaptic membrane.
Since small neurotransmitters are usually docked closer to the synaptical membrane, low frequencies of stimulation are sufficient to increase Ca2+ in this area and release them, without changing the concentration of Ca2+ farther from the membrane, where the bigger neuropeptides are docked. They need therefore higher frequencies (as described above) to be released too. Picture.

If yes, when the highest energy threshold of a vesicle is achieved, does this mean all the other vesicles will release the encapsulated neurotransmitters?

As for the threshold, there has to be enough Ca2+ cations to activate the synaptotagmin, which is why the mechanism described above is working. However, there are few types of synaptotagmins, which have different affinities to Ca2+, which could be a another mechanism, if vesicles containing different neurotransmitter bound different synaptotagmins, but this is just a hypothesis as far as I know, maybe someone will know better here :)

The model in Fig. 3proposes that at least Syts 1, 2, 3, 6, and 7 perform complementary functions in Ca2+-triggered exocytosis whereby Ca2+ binding to each class of synaptotagmins contributes differently to triggering exocytosis

Source


text:
How to predict cancer-related proteins in Protein-Protein Interaction networks
Protein-Protein Interaction networks are known. It's an undirected graph. Each row of the networks is like this (Protein 2 - Protein 6), and It represents the interaction between Protein 2 and Protein 6.
networks:
Protein 2 - Protein 6
Protein 4 - Protein 5
Protein 6 - Protein 5
Protein 5 - Protein 7
...

In this networks, It is known that some of proteins are cancer-related proteins, some are nonCancer-related proteins,
The known proteins:
Protein 4,Cancer
Protein 6,nonCancer
Protein 7,nonCancer
Protein 10,Cancer
...

But the vast majority of proteins is unknown whether is cancer-related protein or noncancer-related protein. How to use these known cancer-related proteins and nonCancer-related proteins to predict the protein whether is or not a cancer-related protein.
I do not know how to solve this problem. I need your help. Thank you very much.


text:
Can a cell start a mitosis if it lacks energy or molecules to complete it?
I'm wondering whether a cell can start a mitosis if it lack molecules or energy to fully complete it. From what I'm reading on wikipedia the cell passes most of its time in the interphase in which the DNA is replicated and then there's the G2 checkpoint to make sure mitosis can start.
However I'm not sure all of this is enough to ensure that the cell has sufficient energy to fully complete the mitosis (is the cell "fueled" in energy and molecules during the mitosis or does it has everything needed before starting it?).
Edit: I'm considering this question for humans in particular, or animals in general if the answer is the same.
When I speak about "molecules" I mean any atom or molecule. In other words, whether the cell needs some material intake from the external environment during the mitosis or not. If not, I consider that it has all the necessary atoms/molecules to fully complete the mitosis without any external need.

When all checkpoints are working, then the most critical is the G1-S. The reason is that once the cell passes this checkpoint, it is committed to either replicate and divide or it will trigger apoptosis. The reason for this is that once DNA replication starts, there is no way for the cell to undo this, so it has to have everything it needs before it commits to DNA replication. There are other checkpoints, however these are to make sure that replication has proceeded properly, or that the daughter cells will receive the exact copies of the genome. AleksandrH's answer provides the basics.

Which organism are you considering? Free-living organisms such as yeast have a variety of mechanisms for sensing nutrients, and regulate proliferation depending on nutrient availability. In animal cells it's very different, as growth factors are the dominant driving forces, and here the role of nutrients as signals is more uncertain / controversial.

I've edited my question to make it clearer (humans basically).

The short answer: no. First, let's get an understanding of the cell cycle control system, as there are some important molecules involved in this system that regulate mitosis. Think of the control system as a series of stoplights: as you mention, there is one stoplight at the G2 phase. There are two additional checkpoints: one at G1 and one in the M (mitotic) phase, just before Anaphase. Why before anaphase? At this stage in mitosis, the cell checks to determine if all of the kinetochores of the duplicated sister chromatids have microtubules attached to them. This is to ensure proper distribution of chromatids in anaphase.
Ok, so back to your question: what are some of the molecules involved, or what are some molecules that the cell might need before it proceeds?

Cyclins - these are proteins that are produced in the late S-G2 phases of Interphase. They activate cyclin dependent kinases (Cdks)
Cyclin Dependent Kinases - as the name suggests, these proteins (kinases) are dependent on cyclin in order to operate. The best known Cdk is called MPF, short for maturation promoting factor.

Throughout the cell cycle, these Cdks, together with their bound cyclins, are able to phosphorylate various proteins in the cell as it undergoes mitosis. For instance, the MPF would phosphorylate lamins, which are proteins on the nuclear envelope that can initiate degradation of the laminar matrix. This is essential in Prophase, when the nucleus is disassembling. There are many other things it can phosphorylate. For a more complete list, look under "Targets of MPF" in the above link for MPF. Take a look at this diagram, taken from Campbell's Biology (9th Ed):

There is one other molecule worth mentioning:

Growth factors - these are proteins specifically released by neighboring cells (relative to the cell that is preparing to undergo mitosis). These are crucial for mitosis to even initiate. A great example of a growth factor is PDGF, which stands for "Platelet Derived Growth Factor". These PDGFs are released by platelets at sites of injury, such as a cut on your knee. The PDGFs stimulate cell division and help your skin to reheal!

There are many other essential nutrients that cells require before they can initiate mitosis; not all have been listed here. Also, I know I didn't necessarily answer the "energy" part of your question, but I hope this helps for your first part.

This isn't the whole story, but it is representative of what would be taught in introductory college level biology.

This answer doesn't really concern energy or nutrients. Cyclins, CDKs and growth factors are all signalling proteins, they merely relay information.

@Roland But the OP didn't ask about nutrients, they asked about energy and molecules, and I covered the molecule part (I mention near the end that I don't know the answer to the "energy" part).

Okay, I guess "molecules" is ambiguous, I would guess in this context it means material / building blocks, not signalling molecules. Perhaps the OP can clarify?

Sure, I wasn't too clear myself because molecules is indeed a very broad term.

I've edited my question, please let me know whether it's clearer now.


text:
How can microarrays be used to quantitatively determine level of gene expression?
This is probably a stupid question because I don't fully understand how microarray experiments work. I'm trying to understand how microarrays work but I'm confused about something which I can't seem to get around without fully understanding how microarrays work, which seems impossible without understanding this.
I understand the principle of how microarrays work. But I don't get how microarrays can tell you how much mRNA is expressed in one kind of tissue vs another kind of tissue. Wouldn't that depend on how much tissue you started with? So if you started with more cells of one kind you will get more mRNA/cDNA and so the intensity of fluorescence at the corresponding spot on the microarray will be greater. So even if the amount of gene expression is lower in one type of tissue the corresponding spot on the microarray might show a greater fluorescence intensity compared to the other type of tissue if more tissue was used.
Also another related question: if you put the mRNA you obtain from cells through PCR will you get the same proportion of mRNA as before PCR? From what I understand PCR can be used to make more mRNA before doing the microarray but because PCR is a random process I'm not sure if it will affect the proportion of mRNA.
Hopefully my questions make sense.

While someone is no doubt putting together an excellent answer, for some good visuals, Google "Microarray presentation" and the first few hits are good powerpoints for microarrays.

Briefly, you can compare different samples, which may have started out as different amounts of tissue, by using control genes. The numbers reported by most standard microarray experiments are not absolutely quantitative (There are 2450 copies of this RNA per cell in this sample), but are rather a proportion as compared to a set of housekeeping genes whose levels are not expected to vary much between the samples you are analyzing.

What about PCRs? do you get the same proportion of mRNA after applying PCR? I would still like an answer to the second question

@MattDMo gave a great answer. For PCR amplification, you are right, this is a random process and while you would expect most RNAs to be similarly amplified, biases can and will occur. Amplification biases are usually assumed to be identical between similar samples and therefore will (mostly) cancel out during the differential analysis.

Housekeeping genes are rarely used in microarray analyses, and there are many issues with finding housekeeping genes by itself  (See https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4430495/ ) .
Models that work with microarrays usually work with making distributions between samples comparable based on the notion that most genes will not be differentially expressed between two conditions. This can involve scaling, or loess normalisation, or the very popular quantile normalisation. RNA-seq analyses oft use library size or upper quartile normalisation to ensure comparability.

From the comments:
MattDMo:

You can compare different samples, which may have started out as
  different amounts of tissue, by using control genes. The numbers
  reported by most standard microarray experiments are not absolutely
  quantitative (There are 2450 copies of this RNA per cell in this
  sample), but are rather a proportion as compared to a set of
  housekeeping genes whose levels are not expected to vary much between
  the samples you are analyzing.

cagliari2005:

For PCR amplification, you are right, this is a random process and
  while you would expect most RNAs to be similarly amplified, biases can
  and will occur. Amplification biases are usually assumed to be
  identical between similar samples and therefore will (mostly) cancel
  out during the differential analysis.


text:
How do place cells switch their response patterns when the subject moves to a different environment?
So I understand that place cells form a map of each environment the subject has been in and responds accordingly in each environment. Question is how do they switch to represent a different environment when the subject moves to a new environment? It seems unlikely that a different set of place cells represent each environment the subject has ever encountered because it seems that would require too many place cells (and since the cell counts in the hippocampus is I presume relatively fixed, "free" place cells run out at some point), so it must be the same place cells "remembering" different response patterns to different environment and responding accordingly in different environments.
From the wikipedia page on place cells: "In a different environment, typically about half the place cells will still have place fields, but these will be in new places unrelated to their former locations."
Muller, R. U.; Kubie, J. L. (1987). "The effects of changes in the environment on the spatial firing of hippocampal complex-spike cells". The Journal of neuroscience : the official journal of the Society for Neuroscience.

Please, to the people who are voting to close: do not vote to close on a question as unclear simply because the question is an area outside of your expertise. It is possible to edit this question slightly to clarify, but this is actually an excellent question and actually provides a fair amount of background information and even includes an external reference.

So I'll try to answer my own question.. Not sure if that's usual but apparently it's allowed.
Ok. I think the question is fundamentally mistaken. Place cells do not "switch" into a different mode when the subject moves to a new environment. Rather, in a new environment, the inputs to these place cells are different, other parts of the brain is telling the hippocampus it is now in a different environment, so naturally, the place cell responds in a different manner. In this sense the place cell is a "computer"  (might be using the word too loosely but hopefully it makes sense), it makes a different output based on information from the senses.
This is my understanding. Hopefully it makes sense.

It is true that individual place cells will fire in many different environments.  However, the exact location where a place cell fires will vary in different environments.  Moreover, two place cells that fire in the same location in one environment could fire in completely different locations in a second environment.  Therefore, the pattern of activity of the network as a whole can be unique for each location in each environment.  The term for this scrambling of the association between place cells and locations in different environments is called "global remapping".
Try: L. L. Colgin, E. I. Moser, M.-B. Moser, Trends Neurosci. 31, 469–77 (2008). for a review.  http://www.cell.com/trends/neurosciences/abstract/S0166-2236(08)00167-7?_returnURL=http%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0166223608001677%3Fshowall%3Dtrue

why the downvote?

Can place cells be seen like the pixels on a screen, in that the same pixels can represent an infinite amount of pictures because it's the pattern of their activation as a group that actually matters, or is the analogy a bad one?

that is a good analogy

Imagine a 2x2 grid. Starting on the bottom left, how many different ways can you move to each box of the grid?
Two times: 1) up, right, down; 2) right, up, left
Now, say each of those combinations are used to store the memory of a different location. In order to activate either sequence, a different input signal would be required to tell the axon to send it's initial signal right, or up.
Your eyes will receive a signal in the form up light, and a specific electrical impulse will be sent down the optic nerve that allows your brain to perceive an image.
There are millions of nerves in your brain, and each combination of impulses serves to store a different memory.
Now, imagine a grid that has a million by a million boxes. How many combinations can you use to fill each box?
And, actually if I recall correctly, the hippocampus is one of the few areas in the brain that is consistently undergoes neurogenesis.

This answer does not attempt to answer the question asked: "how do place cells switch their response patterns in different environments?"

"Different input signals"... ie. from a different combination of signals from, what was my example, the optic nerve.

But how are those signals different? The optic nerve isn't rewiring to connect to different cells in visual cortex when you move from one building to another. You are answering the question "How does a radio tune to different stations?" by saying "Well your radio just listens to different stations depending on what you set the dial": it is essentially just re-stating the question, rather than providing an answer.

Do you think the optic nerve consists of a single neuron?

Absolutely not. I should have been more precise in my comment to say "axons of the optic nerve aren't" rather than the shorthand I used.

Then you tell me, why would the optic nerve only be able to conduct one type of signal? As I said, your eyes collect light - light has many different wavelengths. Does it make sense that collecting one wavelength of light would illicit a different response than would be obtained from a second different wavelength?

Let us continue this discussion in chat.


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What are the dangers related to keeping a human brain alive outside of a human body?
I am interested in learning about the feasibility of keeping a human brain alive outside of a human body.
The question: What obstacles do we need to overcome to be able to keep a human brain alive outside of a human body?
The problems I am taking into consideration are:

connecting nerves with an artificial skeleton, (no need to connect spinal cord?)
delivering signals to the brain to avoid sensory deprivation?,
keeping brain in a "healthy state" as long as possible,
augmenting and stimulating brain,
connecting the brain to external devices without hurting it,
maintaining blood supply,blood transfusion problems (allergic reactions and hives, fever, hemolytic reactions, iron overload etc.)
can a brain be in a sleep mode? keeping brain in a "work" mode,
can a brain function without hormones?,
can we copy a brain?,
can we grow a brain? part of a brain?,
regenerating brain, slowing down the process of brain degeneration,
does the brain need bone marrow to properly function,
maintaining cerebrospinal fluid supply.

I asked this question on Quora, https://www.quora.com/What-must-be-the-conditions-to-keep-a-brain-alive-outside-of-a-body

What dangers are you talking about?

What the dangers (with explanations) related to keeping a human brain alive outside of a human body. I want to understand them better. What are the reasons that we still cannot make a human brain live outside of a human body? I want a "big" answer.


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Is it possible to make bones ossify much slower in adults?
According to Can we artificially increase human growth using HGH,

But during normal human development bones ossify in the process, and in human adults the bones are completely ossified and can't grow in length anymore.

Is it possible to make the ossification process much longer?

Your question title and body don't match. I think you might be misunderstanding the statement. It isn't saying that bone can't ossify in adults and therefore it can't grow, it is saying that because they have already ossified they can't grow. Ossification is basically hardening; completely ossified bone is completely hard and has nowhere to grow from. Ossification can still happen in healing bones in adults after a break, however.

I do understand, but just missed the "not" in the question. Thank you!

Ah yes, that changes the meaning entirely, much better now!

I think what you are asking about is delaying the closure of cartilage growth plates, so that bones continue to grow longitudinally for longer.


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Anaerobic nutrient expenditure
Doing some research into different forms of exercise and energy systems and I realized something odd about the way we talk about energy.
When we eat, we take on macronutrients that are broken down into their smaller components, but on it's face the assertion is that:
Fat = 9 Calories/gram
Carbs = 4 Calories/gram
Protein = 4 Calories/gram

However, when you look at the Krebs cycle, one acetyl sugar molecule generates 32 ATP molecules. Contrast that against Anaerobic metabolism and the Cori cycle, 1 sugar molecule generates 2 molecules (at a cost of 6 ATP in the liver later).
There are all sorts of fitness calculators out there, most of the calculations are based on the Assessment of Physical Activity and Energy Expenditure: An Overview of Objective Measures book, most of which was put together through measuring oxygen in put versus carbon dioxide output (in conjunction with heat radiation.  The clear problem with associating that with weight training and other anaerobic activities is that the other activities don't immediately create CO2 and don't immediately require O2. Some attempts have been made to make estimates based on lactate production in the blood stream, but I'm not confident in those results.
My question is, when there is anaerobic activity taking place, are we actually "burning" more base ingredients than we are exercising aerobically?
In other words, even if I'm only burning 100 Calories lifting weights, would I actually be burning the equivalent of 1,600 Calories based on the glucose I would have converted to ATP aerobially?

The Urea cycle doesn't generate energy and it doesn't break down amino acids. It uses energy to convert ammonia and carbon dioxide to urea. The Cori cycle isn't a metabolic pathway generating energy as you imply. It's a system whereby lactate produced in muscle exercise is goes into the blood and is taken up by the liver, converted back to pyruvate and then to glucose which goes back into the bloodstream. There is no net generation of energy. The liver expends energy so that muscle can use it. You need to sort your question out.

@David Removed the bit about the Urea cycle. I used the Cori Cycle as a shorthand for anaerobic metabolism and the Cori cycle. As stated in the question, not only is there not a net generation of energy, there's a net loss in energy, the muscles create energy at 16 times lower rate than oxidation and the liver expends energy making more glucose, which is the driving factor in the question.


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Do chromosomes uncoil in interphase II?
During interphase II, there is no S phase in which DNA replicates. However, in this stage, do the chromosomes remain wound? Or have they unwound into chromatin form, and recondense during prophase II?

In many cases the chromosomes do undergo some dispersion, they do not reach the extremely extended state of the interphase nucleus, this all happens in telophase -I. It would be correct to refer to the stage between two meiotic divisions as interkinesis which is generally short lived.
The prophase-II is initiated immediately after cytokinesis, usually before the chromosomes have fully elongated.

Interestingly, the very own existence of an interphase II between meiosis I and meiosis II is highly questionable, and a search on Pubmed found an article by Vernet et al., according to which interphase-II exists only in male meiosis.

According to the diagram found on this site, chromosomes do unwind during Interphase II of meiosis.
Sparknotes, although it's not necessarily a reputable scientific source, seems to concur:

Once the nuclear envelope has re-formed after the first meiotic division, the cell enters a short interphase. This interphase is not as specific as mitotic interphase; during meiotic interphase, chromosomes may decondense as the cell waits to proceed with meiosis.

Finally, this website provides a clarification in stating that there is no true "interphase" between meiosis I and II (I guess true interphase would be marked by replication of DNA which, as you state, does not occur):

The period between meiosis I and meiosis II is sometimes called "interphase," this is confusing because it is not a true interphase such as that seen between rounds of mitosis because no synthesis occurs (all chromosomes have two chromatids throughout this stage).


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Do animals get Post Traumatic Stress Disorder after escaping from a life threatening situation out in the wild?
Can animals come down with PTSD in the wild by themselves? For example, would a gazelle that nearly pulls away from a cheetah at the last second and heard the cheetah's jaws clamping down at air right behind it's tail, show symptoms of stress and anxiety after the event?
And if not, why can we as humans succumb to these kinds of disorders, and not animals in the wild?

@YviDe I think he is asking if any level of stress can be emotionally conveyed by an animal in the wild. Any signs of depression after the animal has been exposed to a stressful situation will work, because we cannot so simply measure their emotional stability and talk to them to see how depressed they are as we can with humans.

Peter Levine speaks to this in his book, "Waking the Tiger:  Healing Trauma".  Most animals in the wild which experience a trauma through natural species interactions will go through a sequence of recovery stages.
First, when the trauma or death is considered inevitable, the animal will "faint", losing sensory awareness and consciousness. If the trauma or death is averted, the animal will then partially waken out of that faint, and "shake off" the trauma.
This "shaking off" looks remarkably like an epileptic fit, but when the movements are analyzed, they are a muscle-twitch repeat of the sequence of movements that animal took from the time it realized its hazard to the time it fainted. It is this same muscle-twitch capacity which allows human athletes to practice their skills both mentally and physically, without moving from one spot.
Once the "shaking off" is complete, the animal is able to rise and continue its life activities without any longer-term impacts such as PTSD.
Neurologist Dr. Robert Scaer has an explanation for this lack of PTSD.  Lasting trauma is only taken on when the "victim" experiences repeated episodes of the same kind of helplessness and hopelessness.  We humans are very good at giving ourselves and other species repeated episodes of helplessness and hopelessness, and animals which interact with us often pay a price in PTSD.

In short, yes. It is not the same disorder we as humans suffer from, as our brains are much more advanced and more of our brain is affected. Species of animals such as elephants and chimpanzees, found to have many social and neurobiological similarities with humans, have been found to suffer from this disorder.
PTSD describes anxiety based responses from witnessing life threatening responses. Animals are used quite often when it comes to studying the effects of certain events on a human's life, and many experiments have proved that they do, indeed, suffer from this disorder.
According to this, experiments have been undertaken to replicate torture, loss of a family member, and incarceration humans are exposed to during wartime.
Most elephants in these experiments have suffered from not just PTSD, but from many different stress disorders in captivity. They have been wrenched from their natural environment in which the communities that made up their lives previously are not there to comfort them.
It may be that wild animals can suffer from PTSD for a while, but most likely not for their full lives. Lone animals may suffer from this a little bit more than, say, an elephant in a herd will, so it is entirely possible that they can experience it.

Please add some reference, like for "Elephants have been found to suffer from depression..." or "Captive animals...have been found to suffer from PTSD...".

I just fixed it.


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Genomic DNA isolation from wheat
Can I use dry seed, wheat for example, in place of young leaves for isolation and purification of genomic DNA for PCR amplification?
The goal of my experiment is to validate a novel gene which is either absent or present at a particular chromosome.

That depends a lot on what you want to do in later analysis, especially if you need high molecular weight DNA or not. Please include all relevant details in you question, especially what you already found in your own research. One paper that is very easily found by google would be this one: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3131572/

I want to validate a novel gene which is absent or present at particular chromosome.

Yes sure I tried this and give good result. But you most chose the best protocol and need to be do this manually. I recommend that use CTAB-PVP (which is use 3 buffers EBA EBB SDS %10) extraction method from "Integrated DNA technologies" and if you cant find this I will tell You.


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What Would Happen To a Human Who Died In Space And Stayed There For a Prolonged Period of Time?
Last night I was watching a documentary regarding the Apollo space program, and it spoke about how Apollo 8 orbited the moon 10 times then came back to Earth. It said that while the ship was in orbit no one really knew whether the ship would break orbit or continue to orbit the moon where the astronauts would eventually die.
I thought then that at some point in the future NASA would have maybe recovered the ship and returned them home. If they did this I then wondered what would have happened to the astronauts' bodies during this time? Would they decompose like they would do on Earth, or because of the lack of atmosphere would they not decompose?
There are two scenarios to this. The first scenario is that the ship ran out of oxygen and the astronauts died through asphyxiation. The second scenario would have meant that the astronauts were involved in  a decompression, so that the astronauts were then in a vacuum.

They cannot decompose in the sense of biodegradation but depending on the environmental conditions many other chemical reactions might occur which can break down the molecular constituents of the body. Since there are many possible conditions I guess this question qualifies as broad.

I see your point. But there's only really two environments that they'd be exposed to, one would be carbon dioxide/monoxide rich and the other would be in a vacuum. Both you'd imagine would be extremely cold too.

Not exactly answering your question but this is pretty close.

@WhoElse your link leads to a 404 error page

Title was really good. You may consider it as A man buried in ice for long. An ice where no bacteria are present. It then needs a forensic expert.


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Protein inhibitory process
I usually find substances that activates proteins, like ApoA-I, but I don't find substances that inhibit it.
So in an experimental design, could I consider the absence of a drug that directly acts on the increase of a protein could be considered an inhibitory process?

Your question is not very clear. Can you please rephrase it and add some relevant background?

There are many inhibitors of protein function. Inhibitors are more common, in fact.

No, absence of an stimulating substance is not appropriate to consider as inhibition.
To see why, consider a situation where there are two such substances, substance A and substance B, which can both stimulate protein P (many such relations exist).  If you add only substance B, then protein P will be stimulated. Since P is being stimulated in this case, it would thus not be reasonable to describe the protein being inhibited by the lack of substance A.


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Is the statocyst in Xenoturbella homologous to the statocyst in Cnidaria?
The statocyst is a balance sensory receptor present in both Xenoturbella and in Cnidaria. Since Xenoturbella might be close to the urbilaterian according to some hipothesyses, I was wondering if it is known whether their statocyst organ is homologous (i.e. inherted from a common-ancestor) to the one in Cnidaria?

The statocyst is a balance sensory receptor present in some aquatic invertebrates, including bivalves,[1] cnidarians,[2] ctenophorans,[3] echinoderms,[4] cephalopods,[5][6] crustaceans,[7] and gastropods,[8] A similar structure is also found in Xenoturbella.[9]

So, are the statocyst in Cnidaria and in Xenoturbella homologous (i.e. coded by same or homologous hox genes etc)?

is there a reason why an organ always needs a hox gene coding for it? I know that this is a common mechanism of homology across animals but I'm not sure that it is a general rule, or that any gene can be said to "code for" a structure (as opposed to regulating its morphogenesis).

Hox genes control embryonic development by specifying how different body parts are connected and where. For example, a mutated hox gene in a fruit fly could result in an extra pair of wings, or a missing pair of legs.


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What non-embryological apomorphies do protostomes have?
I've heard they have mostly this:

central nervous system (nerve cord) on the ventral side of the body

Is that shared by both spiralia and ecdysozoans but different from the deuterostomes (where it is dorsal)?
Is there anything else aside from mouth-first vs anus-first?
Ecydosozoans have the cuticle, but it is not shared by spiralia, so while the cuticle is an apomorphy of ecdysozoans, it is not an apomorphy of protostomes.


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What was the morphology of the crustacean common ancestor?
When talking about crustaceans my mental picture is of a lobster or a crab since those are the most iconic crustaceans. This made me wonder what was the morphology of the common ancestor of all crustaceans - did look like a crab or something else?
This is what I found from a cursory google search - https://www.cell.com/current-biology/pdf/S0960-9822(08)00658-1.pdf

While the fossil record does not give a clear picture of the morphology of the ancestor of all crustaceans (the ‘urcrustacean’), it has been argued that it probably would have had a fairly large number of identical or highly similar (homonomous) segments, each of which bore a pair of appendages. This inference of homonomous segmentation in the urcrustacean often leads to description of several extant groups, such as remipedes and branchiopods (brine shrimp, etc.) as ‘primitive’ — or more correctly, having retained ancestral bodyplan characteristics.

Is that correct in the most current understanding - that the urcrusteacan looked like a remipede? Are there any other details that can be inferred about its anatomy or morphology?

Relevant xkcd

There's no consensus as far as I'm aware - there is a deal of argument, including disagreement over the basis of the whole group of crustacea being a real (complete) group or a paraphyletic one. That being said, if xkcd is anything to go by, the common ancestor may be very far back and look nothing much at all  like a crab.

By which I mean, in the link there's an image, some crabs/shrimps etc. appear in the green group, and some things that look very like crabs are in the blue group on the left. Some are in the orange on the right - they all ultimately call back to a single ancestor, but diverged differently. It's more complicated than the diagram suggests and there's more than one common ancestor depending what you count as crustacea.

Would it have looked like a remipede then? Multiple undifferentiated segments, a bit like a millipede or a centipede I guess?


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Did the prostome last common ancestor (LCA) have deuterostomic development (radial cleavage)?
Traditionally bilaterians are divided in protostomes and deuterostomes. However several very basal protostomic clades have strictly deuterostomic development in more recent papers.

Priapulids have deuterostomic development - paper
Arrow worms have deuterostomic development - paper
Nematomorphs might have radial cleavage - paper
Some have suggested that deuterostomy is an ancestral feature of the urbilaterian

Since priapulids might be the most morphologically similar extant species to the ecdysozoan LCA, this might make it very likely that deuterostomic radial cleavage was the ancestral state of the ecdysozoan LCA.
So my questions are:

Is deuterostomy and radial cleavage ancestral to the bilaterian LCA
Is deuterostomy and radial cleavage ancestral to the ecdysozoan LCA
Is protostomy ancestral to the protostome LCA or is deuterostomy its ancestral state, while protostomy is a derived feature of certain sublieages?

Edit: LCA means last common ancestor

Who is LCA?  Especially in titles, abbreviations other than standard chemical ones should be avoided like the plague.

Sorry, I edited my question. LCA means last common ancestor

Thanks. I could explain at length why it’s best to avoid abbreviations, but perhaps I’ll put it on Meta sometime.

There have been phylogenomic arguments that the urbilaterian was deuterostome-like. They go on to argue that protostomes are a well-supported phylogenetic group, unlike the deuterostomes, and protostomy might be the derived feature. They go on to argue that the urecdysozoan was as one might naively expect protostomic, as they would have been a protostome:

If the deuterostomes are paraphyletic, as some analyses suggest, then the last common ancestor of Chordata and Xenambulacraria was the last common ancestor of all Bilateria; Urbilateria would have had the characteristics that have, until now, been used to define deuterostomes—characters common to Xenambulacraria and Chordata would be bilaterian plesiomorphies.

While the evidence for a monophyletic Ecdysozoan group appears to have been somewhat contentious, contemporary evidence for the monophyly of protostomes still seems to be strong. That said, more recent analysis with additional groups treats the Ecdysozoans as monophyletic even if the exact topology of the group is somewhat uncertain. The characteristics of a possible Ecdysozoan LCA are described recently there, where it is treated as definitely protostomic.
The issue at hand seems to be that nematodes have shown rapid molecular and anatomical evolution, which leads to some confusion in character reconstructions in their parent clades. Your Valentine reference (though I haven't read it in depth) is from 1997, when relatively few sequences were available and the C. elegans nematode was one of these few well studied species.
I think that the ancestral-deuterostomy conclusion is supported but that cleavage pattern is no longer considered diagnostic. Happy to be proved wrong on this point- I have not been very successful in finding many pertinent recent references on cleavage as a phylogenetic character. In one of the more recent papers to cite your Valentine paper, they show in fact that the radial-like cleavage of bryozoans (protostomes) as retaining various spiral characteristics. It is therefore plausible that this trait is relatively plastic across metazoans and the apparent radial vs. spiral differentiation may be a bit more complex than the label:

The fate of the early embryonic blastomeres is thought to be causally related to the cleavage pattern during development [2, 163]. In such case, a change in the cleavage geometry would lead to a change in the cell fates. However, the bryozoan cell lineage illustrates a case where the cleavage pattern and blastomere fates are not evolutionary coupled. We find that the fates of the early blastomeres are similar between M. membranacea and spiral-cleaving embryos despite the modified biradial cleavage pattern.

(the 163 reference is Valentine 1997.)

Thank you, and sorry for the delay in responding. One question - at the top of the answer you said "arguing that the urecdysozoan was as one might naively expect, protostomic, and protostomy might be the derived feature:" - did you mean protostomic or deuterostomic there?

@sashoalm tried to clarify- yeah, I wrote that confusingly. Basically, protostomy is derived relative to deuterostomy, and protostomes form a pretty good clade. Based on that, since the urecdysozoan is a protostomes in a well supported protostome clade, it makes a lot of sense that it would be a protostome.

btw I also asked a narrower question specifically for the urecdysozoan at https://biology.stackexchange.com/q/115104/23100 since it probably can be answered more definitively while this one is a bit more broad and harder to answer.

@sashoalm Hm, the comments on that question make me wonder whether my answer is wrong. Notably it looks like there may be a anatomical vs. molecular phylogenetic discordance here.

Yes, when I talk about protostomes, it can be a bit ambiguous because I have to distinguish between protostomes (the clades) and "protostomy" - the idea of "mouth-first" vs "anus-first" embryonic development. I don't doubt the protostome clade itself (that is spiralia+ecdysozoa), but I was wondering if the ancestral state of both nephrozoa (that is protostomia+deuterostomia) and also protostomia is "anus-first" and radial cleavage.


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Was the Ecdysozoan last common ancestor a priapulid-like organism?
Is it possible that the LCA of all ecdysozoans was priapuilid-like, or very similar morphologically to priapulids, while all the other body plans - arthropods, lobopodians etc were more derived?
Based on the following:

T. Pedum ichnofossil which marks the Cambrian boundary and is described to be similar to traces made by modern priapulid worms
Genetically, scalidophorans are described as sister lineage to all other ecdysozoans
Very early Cambrian scalidophoran worms look almost identical to modern priapulids
This rather insightful video which suggests that the ancestral mouth of arthropods was radiodont-like - circular with teeth similar to a priapulid
Molecular clock studies have revealed that unlike arthropods or nematodes priapulids are slow-evolving and their mitochondrial gene order may be unchanged since the Cambrian.

I guess my question is - morphologically, is the LCA of all ecdysozoans more likely to look like a modern priapulid than a modern insect?

Your intuition is close to what I think is the consensus in the field: that the ecdysozoan LCA had a terminal mouth, worm-like appearance, pseudocoel, maybe scalid-like structures and lacked true segmentation, I'd have to check this against the current literature.

@alephreish And it would have had ecdysis (molting)) as well, right?

Sure. I've posted my answer now.

Indeed, reconstruction of the appearance of the last common ancestor of the crown-group Ecdysozoa which covers the three major clades (extant representatives in parentheses):

Scalidophora (modern priapulids, kinorhychs and loriciferans)
Nematoida (nematodes and nematomorphs)
Panarthropoda (tardigrades, onychophorans, arthropods)

is uncontroversial thanks to a large degree to the fact that the phylogenetic relationships between them have been resolved with phylogenomics: Scalidophora + (Nematoida + Panarthropoda). Position of Loricifera accroding to molecular data has been controversial but the (morphologically transparent) monophyly of Scalidophora is now largely established. The ancestral morphological characteristics of the LCA (not necessarily synapomorphies) of the crown-group are:

ecdysis
pseudocoel
circum-oral brain
terminal mouth
protonephridia
digestive system opening at both ends
vermiform annulated but unsegmented body
pharyngeal armour (cf. priapulid pharyngeal teeth and various putatively homologous structures across those ecdysozoans that have them)
maybe: circum-oral armour (cf. scalids) although homologization is not certain across the different taxa
proboscis but maybe not introvert covered with scalids -- notice however that introvert is in fact present in scalidophorans and nematomorph larvae
maybe: a mouth cone which is present in scalidophorans, nematomorph larvae and tardigrades

One of the open questions is the position of the several priapulid-like fossils (see one of the attempted reconstructions here). One of the the most prominent and better studied taxa among them are palaeoscolecids which have an armoured introvert, a long nematoid-like body and cuticular plates which some see as homologous to cuticular plates of Cambrian lobopods. They might turn out to be stem-group priapulids, stem-group scalidophorans or maybe they diverged even before.
Reconstruction of the ancestral body plan of the total-group Ecdysozoa (i.e. including taxa diverging before the crown group) is more problematic as it depends a lot on the interpretation on the morophological features and position of some critical fossils. For example, Acosmia, has been demonstrated to lack a true introvert and pharyngeal armature which lowers the probability of these structures to be present in the LCA of total-group Ecdysozoa. Analogously other, such "priapulid-like" forms as Ancalagon and Fieldia which might belong to stem-group Ecdysozoa have a non-retractable proboscis as well.
Finally, the alien-looking Saccorhytida have been proposed to be sister group to (the rest of?) Ecdysozoa which has a profound effect on the reconstruction of the ancestral body plan of the group but the evidence that they belong to Ecdysozoa is controversial.
To answer your question directly, whether the LCA of Ecdysozoa was priapulid-like: not exactly. It must have had a proboscis but maybe not an introvert (this depends on whether the introvert of nematomorph larvae is a remnant of the same structure as in scalidophorans and whether we are talking about crown-group or total-group Ecdysozoa), certainly not an introvert armoured with scalids which is a synapomorphy of Scalidophora.

Thank you! I didn't know about Acosmia maotiania from the article you linked - https://bmcecolevol.biomedcentral.com/articles/10.1186/s12862-020-01720-6, but it sounds very interesting. My guess now is that Acosmia maotiania was either the urecdysozoan or as closely related to it as you can get.

Yes, it does look like this (alongside Ancalagon and Fieldia). Notice that I've corrected the points concerning introvert and mouth cone in the crown-group Ecdysozoa. I'll refresh my memories on these points and add more details and citations later. The situation with the circum-oral armour is also quite complicated.

Thank you, and I have added one more question about deuterostomic development (radial cleavage) in the ecdysozoan LCA - https://biology.stackexchange.com/q/115104/23100


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Barcode exchange between neighbouring cells
I am currently interested in equipping animal cells in a tissue with individual barcodes. These barcodes should get amplified in the cells (not to a cell degrading extent, of course), and also transmitted to neighbouring cells.
The question is now, how do I a) insert these barcodes into the cells and b) how do I transmit them to the neighbouring cells.
In the Zador et al. paper "Sequencing the connectome" (http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1001411), it was proposed to use a transsynaptic virus like as rabies or pseudorabies, for making that exchange possible between neurons that are synaptically linked. Here, however, I am interested in the directly neighbouring cells and I am insecure whether it makes sense to use transsynaptic virus here.
If so, it would be great, but if not, do you know some alternatives?