text:
Seed Dormancy and Seed Transportation
My textbook says "As the seed matures it loses water and enter dormancy". I am confused as to whether this happens before the seeds are transported or after, and as to how seeds know to become dormant when required and then become active again.

I presume what the author is trying to convey is that as a seed ages, it enters a quiescent phase as a result of natural desiccation. Without more context, I'd guess that the quote you've provided here is referring to secondary dormancy, which is a response to unfavorable germination conditions. By definition, secondary dormancy occurs after the seed is released from the plant.
The mechanisms of secondary dormancy are not well understood, and resumption of embryonic function could be dependent on any number of factors. Generally, dormancy continues until such time as favorable conditions are observed by the dormant seed. For Avena sativa, seeds become dormant if temperatures in excess of the maximum for germination are observed. Other seeds become dormant in response to over- or dehydration, temperature variability, age, other factors, and combinations ad nauseum.
Some of the more common environmental causes of exiting dormancy include afterripening, chilling and lighting.

So as a seed matures it becomes dormant, and is than released and transported. Eventually once it ends up in a favorable habitat it becomes active again

It happens that way in primary dormancy. What I described above was secondary dormancy, in which release and (potentially) transportation occur prior to dormancy. Primary dormancy usually ends the in a similar manner as with secondary dormancy, but the seed is released and transported in an already-dormant state. Reading over the textbook quote again, it could well have been referring to primary dormancy.

I'd just like to say how happy it makes me to see primary and secondary dormancy being discussed on bio.SE: very happy indeed :). Also: this is exactly the answer I would have given.


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How is nutrient uptake in humans measured?
There are many claims made about nutrient absorption, eg "Juicing helps you absorb all the nutrients from the vegetables".  I'm curious to know how nutrient uptake is measured.
Eg is it a pure nutrients in vs nutrients out equation?  Or are blood levels of nutrients measured before and after consumption?
What ever the methods, are they reliable enough to support claims made about relative absorption rates of certain diets or eating practices?

Depends on the nutrient type. Typically you would need an distinct assay or a molecular sensor for each nutrient type.

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Why does adding calcium chloride aid in bacteriophage infection?
We are attempting to isolate bacteriophages from environmental soil samples and most protocols recommend adding CaCl2 to the media to aid bacteriophage infection, even though they all recommend different concentrations.
This paper claims:

Addition of divalent ions (CaCl2 and MgCl2) may also facilitate plaque formation through aiding phage adsorption to the bacterial receptor

But how does CaCl2 aid phage adsorption exactly? And by extension, is there an ideal concentration of CaCl2 that maximizes the adsorption when growing bacteria in liquid media?

Not knowing anything about phage infection itself in this context, I would guess that the ideal concentration varies depending on phage, host, and environment. There may not be a universal ("You should always use 10 mM") ideal concentration. If the papers you're reading are all dealing with the same context you are, and the recommended concentrations aren't too far away from each other, i.e., over an order of magnitude or so, then averaging them might not be a bad idea. Or, pick one paper and use its conditions as your guide so you can replicate their results.

@MattDMo we are  going to test with two concentrations since the concentration varied so much in the various papers. 1 mM/liter and 50 mM/liter. We did read one paper that seems to use 3 M/liter which seems high. None of the papers detail what mechanism makes CaCl work however.

@JohanWikström doing a range of concentrations would be a good idea - you'll likely select different populations of phage. 3M may have been a typo as I don't think there are any bacteria beyond extreme halophiles that will tolerate that sort of salt concentration. I would guess cofactor - divalent cations are commonly cofactors for proteins.

The relationship between calcium ion concentration and efficiency of phage infection has been observed since Delbrück's work classifying phage mutants.1 A short letter in Nature authored by H. Williams Smith in 1948 is the first source I can find recommending CaCl2 supplementation of media for bacteriophage propagation.2

Stassano and de Beaufort, Asheshov, Bordet, Burnet, and others have reported on the inhibitory effect of sodium citrate when added to media used for the demonstration of phage action. This suggested that a deficiency of calcium in the medium might be responsible for the inhibition of phage action referred to above. On the suggestion of Dr. G. P. Gladstone of the Lister Institute, calcium chloride in concentrations of $M$/100 were added to two of the batches of inhibitory media; the third was unfortunately discarded. The effect was striking. The media were now found to be completely satisfactory for the demonstration of bacteriophage activity.
As a result of this experience, it is considered advisable to add calcium chloride to solid culture media that are to be used for the typing of staphylococci by the bacteriophage method.

To answer your first question ...

But how does CaCl2 aid phage adsorption exactly?

... structural and biochemical studies have shown that calcium ions are important cofactors for many adhesion-related phage proteins. Some examples:

p2, a phage infecting Lactococcus lactis, requires Ca2+ to induce the "open" conformation of the receptor binding component of its baseplate.3
Bacillus phage SPP1 requires Ca2+ to stabilize the infective conformation of its tail tip.4
Escherichia phage Mu needs Ca2+ for efficient binding of its central spike to the host membrane.5

Beyond adsorption, Ca2+ has a role as a counterion during phage DNA injection 6-8 and as a stabilizing factor during capsid assembly.9,10

To address your second question ...

is there an ideal concentration of CaCl2 that maximizes the adsorption when growing bacteria in liquid media?

... that will depend on the phage-bacteria system and other details of your culture environment.

References

Delbrück M. Biochemical Mutants of Bacterial Viruses. J Bacteriol. 1948 Jul; 56(1):1-16.
Smith HW. Calcium-deficient media; their effect on phage action. Nature. 1948 Mar 13; 161(4089):397.
Sciara G, Bebeacua C, Bron P, Tremblay D, Ortiz-Lombardia M, Lichière J, van Heel M, Campanacci V, Moineau S, Cambillau C. Structure of lactococcal phage p2 baseplate and its mechanism of activation. Proc Natl Acad Sci USA. 2010 Apr 13;107(15):6852-7.
Goulet A, Lai-Kee-Him J, Veesler D, Auzat I, Robin G, Shepherd DA, Ashcroft AE, Richard E, Lichière J, Tavares P, Cambillau C, Bron P. The opening of the SPP1 bacteriophage tail, a prevalent mechanism in Gram-positive-infecting siphophages. J Biol Chem. 2011 Jul 15;286(28):25397-405.
Harada K, Yamashita E, Nakagawa A, Miyafusa T, Tsumoto K, Ueno T, Toyama Y, Takeda S. Crystal structure of the C-terminal domain of Mu phage central spike and functions of bound calcium ion. Biochim Biophys Acta. 2013 Jan;1834(1):284-91.
Fernandes S, Labarde A, Baptista C, Jakutytè L, Tavares P, São-José C. A non-invasive method for studying viral DNA delivery to bacteria reveals key requirements for phage SPP1 DNA entry in Bacillus subtilis cells. Virology. 2016 Aug;495:79-91.
Alatossava T, Jütte H, Seiler H. Transmembrane cation movements during infection of Lactobacillus lactis by bacteriophage LL-H. J Gen Virol. 1987 Jun;68 (Pt 6):1525-32.
Cvirkaite-Krupovic V, Krupovic M, Daugelavicius R, Bamford DH. Calcium ion-dependent entry of the membrane-containing bacteriophage PM2 into its Pseudoalteromonas host. Virology. 2010 Sep 15;405(1):120-8.
McKenna R, Bowman BR, Ilag LL, Rossmann MG, Fane BA. Atomic structure of the degraded procapsid particle of the bacteriophage G4: induced structural changes in the presence of calcium ions and functional implications. J Mol Biol. 1996 Mar 8;256(4):736-50.
Plevka P, Kazaks A, Voronkova T, Kotelovica S, Dishlers A, Liljas L, Tars K. The structure of bacteriophage phiCb5 reveals a role of the RNA genome and metal ions in particle stability and assembly. J Mol Biol. 2009 Aug 21;391(3):635-47.

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How are some exotoxins heat-stable, despite being proteins?
While studying microbiology, I came across bacterial exotoxins and endotoxins. It's understandable that endotoxins are lipopolysaccharides and hence heat stable.
Exotoxins are proteins and hence heat labile. But there are few exotoxins which are heat stable.
For example, Bacillus cereus produces two types of toxins that are involved in food poisoning— emetic toxin (heat stable preformed toxin) and diarrheal toxin (heat labile).
I don't fully understand the reasoning but I have a thought that, the heat stable exotoxins are small proteins/peptides which after denaturation (by heat) can refold back properly.
If anyone would help, it would be great!

Why do you think that these proteins should be heat stable?

Like I mentioned in the question, I think heat would destabilize the protein folding, but small proteins might fold back...just saying

@ANandiΨ The construction "Why few..." in your title implies "Why are there not many?" But the body of your question suggests that you want to know "How are some exotoxins heat stable"? I'll make an edit, but feel free to roll back if not correct.

@ANandiΨ A priori, unless heat-stability is being selected for, one would expect that most proteins would not be head stable.

@acvill the edit is helpful... thanks

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Why does ionizing radiation cause only DNA double strand breaks?
It's known that ionizing radiation such as X-ray and $\gamma$-ray, cause DNA damage, specifically double strand break. Why is it so? I mean, why not single strand break, why not pyrimidine dimer?

Ionizing radiation does cause single-strand breaks and other kinds of DNA lesions that are not double-strand breaks.  However, double-strand breaks are the most difficult to repair and are thus the most likely DNA damage type to result in mutation and / or cell death.  See Fig 1. from DNA damage response signaling pathways and targets for radiotherapy sensitization in cancer:

DNA damage induced by ionizing radiation. The major types of DNA damage induced by IR include base and sugar damage, single-strand breaks, double-strand breaks, clustered DNA damage, and covalent intrastrand or interstrand crosslinking


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Why does a wound itch while it's healing?
During healing of wound (almost in final stage) it begins to itch a lot around wound.
I wondering what the reason for this is?

While healing your wound the cells in and around it are enhancing their communication via messenger substances to quicken and coordinate the healing process. These messengers irritate the sore nerves which we feel as itching.
Skin tension is another factor to itching.
Scratching is bad for the healing process in any way.

Interesting answer. Could you provide a reference?

@GoodGravy i don't remember the exact source but this i found on a quick search.

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Question on compatibility of blood groups
How come a person with blood group O can donate to a person with blood group AB? Since there are A and B antibodies in the O blood group blood surely this would cause agglutination in the blood of the recipient?
My first hypothesis was that the antibodies and red blood cells were separated through use of a centrifuge or something but blood transfusion has been used for hundreds of years before this machine was even invented? What is going on here? Or was it just that before blood types were discovered only people with identical blood groups could donate/recieve from one another?

Yes, the recipient's red blood cells would be attacked by the donor's blood. However, there are not that many antibodies in the donor blood that would pose a major threat - and the donation is diluted by the many liters of recipient blood! Here is a complete answer, the part at the bottom is relevant to your question.

Good question! It just so happens we already have an answer for it! (See the above links, and thanks to @SPr for identifying it.


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Doesn't pasteurization kill gut bacteria such as Akkermansia muciniphila?
I've recently watched two presentations on YouTube (this and this) about Akkermansia municiphila, a commensal bacteria that feeds on the mucus of the gut.
What I find confusing is their claim about stabilizing Akkermansia and making it commercially available via pasteurization.
How can it be used as a priobiotic if they pasterize it? Doesn't that make it inactive?

This is interesting since Akkermansia muciniphila is a strict anaerobic and does not produces spores (which was my first thought on it). However, digging up the original publication (see reference 1) solved the question:

Unexpectedly, we discovered that pasteurization of A. muciniphila
enhanced its capacity to reduce fat mass development, insulin
resistance and dyslipidemia in mice. These improvements were notably
associated with a modulation of the host urinary metabolomics profile
and intestinal energy absorption. We demonstrated that Amuc_1100, a
specific protein isolated from the outer membrane of A. muciniphila,
interacts with Toll-like receptor 2, is stable at temperatures used
for pasteurization, improves the gut barrier and partly recapitulates
the beneficial effects of the bacterium.

The effect on the experimental animals by these bacteria seems to be mediated by the Amuc_1100 protein which interacts with the Toll like receptor 2 and is responsible for bacteria-host interactions. It turned out that the protein is stable at the temperatures used for pasteurization.
The pasteurized bacteria seems to influence the energy expenditure and decreased the the food efficency (see reference 2):

We confirmed that daily oral administration of pasteurized A.
muciniphila alleviated diet-induced obesity and decreased food energy
efficiency. We found that this effect was associated with an increase
in energy expenditure and spontaneous physical activity.

However why this effect can be seen with the pasteurized bacteria still seems unknown (at least I haven't found a scientific article yet).
References:

A purified membrane protein from Akkermansia muciniphila or the
pasteurized bacterium improves metabolism in obese and diabetic mice

Pasteurized Akkermansia muciniphila increases whole-body energy
expenditure and fecal energy excretion in diet-induced obese mice


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Do symptoms indicate 'strength' of someone's immune system?
(I am a layman, forgive any misconceptions)
A friend recently got a cold; she had a sniffle, a sick feeling and it was barely more than an annoyance.
A week later I get - presumably - the same cold; I get temperature and lethargy, plus the usual cold features such as sore throat, and spend a few days in bed.
To illustrate the point further: most times I get a cold I feel terrible; have a fever, lethargy etc. I will spend various days in bed hoping for it to go away.
My friend on the other hand doesn't even understand what a fever feels like. For her, colds just make her 'sneeze more'.
Who is said to have the stronger immune system?
Is it that her immune system is 'strong' enough to fight off all viruses easily without breaking a sweat? Or does it imply her immune response is weaker, producing less symptoms than mine?
Or is saying that one of us has a 'stronger' immune system a gross oversimplification?

A person is said to have a weak immune system when he/she gets infected very frequently. Such individuals tend to get more tired than others, their wounds heal way slower than others.
You may want to read more about weak immune systems here.
Different symptoms or duration of infection don't decide the immunity status of any individual.
Every human body has its own way to deal with pathogens. A body that has previously encountered a certain pathogen will behave differently to the body that is encountering the same pathogen for the first time. This usually happens because the antibodies pool differs from person to person.
Another reason can be the type of pathogen variant. The seasonal cold which seems the same for two people might be actually different. This happens because pathogens (bacteria, viruses) mutate themselves over generations. The strain might vary between two individuals.
Another reason can be the lifestyle also. A person who workouts frequently may show different symptoms of any infection when compared to a person who has a sedentary lifestyle. The more you exercise the stronger your immune system gets.


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If the flu vaccine is the exact same as last year, would it still offer protection this year?
I understand why the flu shot changes and that's why we get an annual vaccine; however, if the vaccine is the same as last year, wouldn't people theoretically still have the same antibodies this year? Do antibodies disappear after a year?

If one flu strain wouldn't change between the years (meaning there wouldn't be any new mutations in it) and the same strain would circulate again in the following year, the "old" vaccination would protect people. This is the same as with other viruses which do not change, once you reached a protection (usually one basic immunization and one booster), this protects for a long time (see measles vaccination as an example here).
There are surprisingly little articles available on how long the immune response after a vaccination lasts. There is a difference between the livettenuated influenza vaccine (LAIV) and the inactivated influenza virus (IIV), with the former giving a less good protection (see reference 1).
Data for the LAIV suggests that there is protection for at least one year after the vaccination (see reference 2).
However, there is evidence that the immunity against the flu after an infection against this specific strain might last life-long, as a paper demonstrated on survivors of the 1918 flu epidemic (see reference 3).
Having the flu against being vaccinated against it is a complete different thing (higher exposure, probably more antigens), but the question is if the immune response against vaccinations may also for a longer time.
The problem with the flu vaccination are:

The strain circulating in a year is changing.
The stains are mutating, making them different from the virus used for vaccine production. Depending on how different these mutations make the circulating strain, it can render the vaccine more or less useless. This is something what happened this flu season.
The flu vaccine is prepared from different flu strains to cover a broader range. In a tri-valent vaccine these are typically 2 Influenza A and one Influenza B virus strains, in tetra-valent vaccines 2 A and 2 B strains. The composition is based on a prediction (from epidimiologic data) which strains are likely to circulate in the next season and is done early in the year for the following season (production takes time, etc.).

Because of these problems, researchers are working on a universal flu vaccine; which is based on virus parts which do not change and should protect against all virus strains. Then only the basic vaccination would be necessary as with other vaccinations.
With all these limitations, it is still advisable to get a flu vaccination, as it is a pretty severe infection.
When not challenged (i.e. upon infections) antibodies are cleared from the blood within a few weeks, but there are low amounts produced (which are enough for detection). The immunologic memory is maintained by memory B cells which can ramp up production of the highly specific antibodies in a very short time - this is the reason for the protection by immunizations.
References:

Modest Waning of Influenza Vaccine Efficacy and Antibody Titers
During the 2007-2008 Influenza Season.
Longevity of B-cell and T-cell responses after live attenuated
influenza vaccination in children.
Neutralizing antibodies derived from the B cells of 1918 influenza
pandemic survivors

Thank you very much. It is concerning as there have been many children dying from the B strain, that experts are claiming is the one covered in the vaccine. The vaccine is exactly the same as last year, so wondering if those who received the vaccine last year would have some protection, obviously taking into account changes and mutations

The majority of deaths usually occurs in the non vaccinated.

Chris, can you support your claim of prolonged immunity against influenza vaccine (i.e. antibody persistence)? Some viruses do have astounding antibody persistence. Others have little.

@anongoodnurse Yes, there are some papers, I will dig them out. There was especially one paper on survivors of the 1918 flu. It has to wait, until I have paper access again at work.

So in saying this. Antibodies that have developed after receiving the flu vaccine does not wear off after a year, correct? So someone that received the shot in Canada last year but not this year, would have the same level of antibodies, as someone who received it this year. As the vaccine has the exact same strains included in the shot as last year. Is this correct


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What are the meaning the nomenclature in high cell density cultivation for the production of a protein?
I'd like to know the meaning of these terms in a HCDC studies of a modified strain.
Max specific yield (mg/g DCW)
Max volumetric product yield (g/l)
Specific product formation rate Qp (mg/g DCW/h)

I wanted only the definitions of each

Maximum specific yield = milligram product per gram dry cell weight (DCW).
Maximum volumetric yield = gram product per liter culture.
Specific product formation rate Qp = milligram product per gram dry cell weight per hour.


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Why the genome is divided into several chromosomes and not just a single big chromosome?
In many eukaryote species, there are several chromosomes. In humans, for example, there are 23 pairs of chromosomes.
Why are there several chromosomes and not just a join of all chromosomes into a single big chromosome?

What do you mean by the human chromosome? There are 23 pairs of chromosome in the human genome. What do you mean by chunks? Sounds like instead of "chunks", you meant "chromosome" and instead of "chromosome", you meant "genome". But then the question "why" is more than obvious. There are 23 pairs of chromosomes. There is no arbitrary decision to be made here. We just counted them. Can you please clarify your question?

After edit: Is your question why are there 23 small(-ish) chromosomes and not just a single big chromosome? If yes, is your question specific to humans or to any life forms (or any eukaryote at least) that have more that one chromosome?

Yes. why are there 23 small(-ish) chromosomes and not just a single big chromosome?. And my question is specific to humans

I rewrote your post trying to make it on-topic. Please feel free to roll back if you don't like the edit. I removed the reference to information entropy because really I don't know what you had in mind there and you would weirdly restrict the space of possible reasons by limiting the field of studies that can explain such phenomenon.

I think the answer is related to Information theory

Can you explain your thoughts for why you think that? Again, please feel free to re-edit your post as you like. You don't have to keep my edit.

Remi.b I appreciate a lot your help, and I will not re- edit your post, thank you. You are very helpful

I think the answer is related to Information theory because the genome system has : 1- redundancy by doing chomosome pairing. 2- Error Detection and error correction by base pairing (A - T & C - G)

@ManuelMilla —   This is better included in a revision of your question. Many people do not read the comments and they are not formally part of the question.

please add a paragraph that goes something like that "Based on sources (1,2,3) I think that's because of (reason 1,2,3,4). Is that correct? I dont understand why (2,3) are important" or something. In other words, show your work

@David -- When you ask for a revision in the question, Do you mean to add the reference to my conjecture about the relation between the Information theory and the genomic code?

@aaaaaa - I understand you. I will prepare the possible answer, and when it is ready I will edit the question... I will just add my conjecture at the end of the question

@ManuelMilla I think you probably know too little about both information theory and genetics to attempt relating them. There is no relevance to information theory in your question and although you listed two possible things you have not made any logical link between them and your question.

@Bryan Krause - Imagination is more important than knowledge. Albert Einstein. I have a Degree in Information systems and a specialization in Data mining and knowledge discovery. For me, asking questions is more important than answering questions. I am ignorant in many fields of the knowledge but I use my imagination in order to make questions

I agree that asking questions is important, but tags are here to help people find questions/answers appropriate to their interests; tagging a post with tags that are not relevant is not helpful.

"For me, asking questions is more important than answering questions." Fine, but that's not what SE Biology is about. It's about providing precise answers to answerable biological questions. Questions that only allow opinion, speculation or discussion belong elsewhere.

If you answer your own question (which is perfectly fine) please use the answer section. And please do it in english, as this is the language of this community. It is also not the first language of most persons here. I edited the question accordingly.

Does this answer your question? What limits chromosomal length?

The leading contender of "why" in my former lab, is that if a chromosome becomes too long, a cell cannot fully isolate the chromatid to a daughter cell. Ie in a giant chromosome, the chromosome arms trail so far behind centromere, that the arms are of two sister chromosome are still touching each other even through the centromere have reached the opposite poles of a dividing cell. This prevents nuclear reformation. It would also mean, that the size of the cell will determine the chromosome maximum size.
At present this is a hypothesis. We have only just created the method to make ultra large synthetic chromosomes. I expected an answer is 3-5 years (2021-2023). However we say there is nothing special to a chromosome number.

Related to your answer: https://biology.stackexchange.com/a/35150/6307

So your answer is "We don't know, but a plausable hypothesis is...". Possibly better as a comment.

@canadianer — a very pertinent reference. Perhaps the question should be closed as a duplicate.

Was a bit long to put in to the comments. Right now, this question has no answer, only a hypothesis. However the tools to answer it a systematic manner has been developed.

Short Answer: Humans reproduce sexually and evolution, with some random effects, has led to 23 pairs of chromosomes.
Explained:
Reproduce Sexually - Bacteria do have one large piece of DNA.  This works for bacteria because they will copy all their DNA, and split the two copies creating two "clones" of each other.  Humans, on the other hand, half the amount of genetic material in their sex cells (sperm and egg), which when combined together in a zygote sums to the full or normal amount of DNA.  This is why nearly every cell in the human body has 23 pairs, or 46 total chromosomes.  To have sexual reproduction we therefore need at least 2 chromosomes in the normal, or diploid, cells.
Randomness - While evolution is often taught as a very directed process, there is actually a great deal of randomness that shift outcomes.  The best example is that apes, our closes descendent have 24 chromosomes.  In becoming human, two of theses chromosmes somewhat randomly fused together into the human chromosome 2.  While there could be some selection advantage, what it would be is unclear.  Similarly, many other species have a variety of chromosome number.  Biologists have been unable to directly relate a cause for the number of chromosomes in each species (see UCSB and Museum of Innovation), and have therefore determined that the number is mostly based on random effects.  A good metaphor is that a chromosome is a bookshelf and genes books, the character of a library is not defined by the number of bookshelves or books, but rather the contents of the books.
Evolution - Chromosomes fill several important roles in a cell.  Firstly, during normal cell life they unravel partially and gain/lose different markers (histone marks), which can direct different proteins as to which genes should be transcribed. Secondly, during mitosis the chromosomes must become very compact, and split exactly along the mitotic plane, and during meiosis the chromosomes crossover, exchanging DNA with the other pair.  The mechanics of each event is inherently based on the size and number of chromosome.  For all of these roles, the number of chromosomes clearly has some impact, although the exact relationship is unclear.  So while evolution may select for an optimal number, the strength of the selection is likely very weak and overpowered by the random effects.

So you do not actually have an explanation. You just say that meiosis and mitosis are complicated so that it’s best for the DNA not to be too long, the only evidence for which is the circular argument that there are multiple chromosomes.

Answer has been edited.  The key key answer is that the number of chromosomes must be greater than 1, after that it is mostly random as to how many.  Although I tried to indicate that since the number of chromosome does change the mechanics of the cell, it would be a property evolution could and therefore likely has selected for.

@David: You need to remember that this is evolution, not "intelligent" design.  There might be no real reason, other than it happened to work well enough for the organisms having multiple chromosomes to survive & reproduce.  You might note that in plant breeding, at least, it's quite common to create duplicate chromosomes (polyploidy: https://en.wikipedia.org/wiki/Polyploid )  Once the duplicate chromosomes exist, they can differentiate, creating more space for genetic diversity.

@jamesqf — I found your comment puzzling, so I googled "intelligent design". It appears to be some religious superstition of the native peoples of North America in West Virginia and the like. I live in Northern Europe, in an atmosphere of scientific enlightenment. This taught me to beware of people who substituted scientific dogma for religious dogma and argued that something was so because it was so. It is possible to say "we don't yet know" without implying that it must be the work of some "god".

So your key argument seems to be that for sexual reproduction there needs to be "at least two chromosomes", in humans X and Y. But this argument does not hold because there are other animals in which sex is determined differently, e.g. in alligators, by temperature. And please do not preach to the poster. Most of your answer is patronizing biological background, rather than considered argument, let alone evidence.

@David 1. I think you are confusing sex determination with sex reproduction. Sexual Reproduction requires a 2n cell (n being number of chromosomes) becoming a n cell in meiosis.  Therefore, the total number of chromosomes must be at least 2, with n=1.  2. Intelligent design is not a West Virginia thing, but an idea at least exposed to students in many states. 3. I don't know how I am preaching

But n can = 1. (My other remark was a reply to @jamesqf.)

@David: My point is that when you ask "why", the only answer is often that it just happened that way.  If a few random chances fell out differently, life might be based on single chromosomes (or a single pair).  Now you might instead ask whether it's likely that a single long chromosome might break, whether by chance some might break in ways that allowed the organism to survive, and whether there are might be advantages to multiple chromosomes that allowed these broken-chromosome organisms to eventually become the norm.  But that's hindsight :-)

@jamesqf — In my opinion the key point is when you ask why. Often, as with the interminable evolution questions, the answer is never. It is clear that there is no way of knowing why we have five fingers instead of six etc. — we appear to agree on that point. In such cases it is better not to ask, and if the question is asked, not to answer. However for this question there probably is an answer, and there may even be experiments or an evolutionary paper trail that would support or reject the an idea such as maximum viable length. None has been yet been presented here unfortunately.


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Why does T-cell Cancer Therapy require a large tumor mutanome?
An article I read about Neuroblastoma states that the fact that Neuroblstoma has a small mutanome means that it is not viable to apply the classic T-cell immunotherapy. Why is this so?
The article can be found here: Neuroblastoma: Developmental Biology, Cancer Genomics, and Immunotherapy

The article is also available on PubMedCentral.

Did you get a chance to look at the article @MattDMo?

I changed the link to the article, the authors manuscript is available through PubmedCentral.

Understanding the link between the size of the tumor mutanome and its immunogenicity requires a basic understanding of T-cell development and regulation.
When normal human cells degrade proteins via the proteasome, some fragments of these proteins, called peptides, are bound to specialized cell-surface proteins called MHC. T-cells express a specialized receptor, called the TCR, which recognizes peptides bound by MHC proteins on other cells. The specificity of the TCR, what peptide/MHC combinations a T-cell can recognize, is randomly generated by targeted mutations and VDJ recombination in the TCR genes during a T-cells development. Each clonal population of T-cells (T-cells derived from the same precursor) have a unique TCR that recognizing different peptide/MHC pairs. When a T-cell's TCRs recognize a peptide/MHC combo on another cell, the T-cell is activated.
Because the TCR is generated randomly, many TCRs in T-cell precursors can recognize peptides derived from normal human proteins. To prevent these precursors from becoming T-cells that can attack the body, they are regulated in several ways. Many self-peptide recognizing precursors are destroyed in development, others are converted into regulatory T-cells that instead produce anti-inflammatory cytokines that tune down immune responses. Those those that escape these regulatory processes are later inactivated by other means.
What does this have to do with cancer? Well, most of a tumor's proteins are normal human proteins, and the human body does its best to suppress immune responses against normal human proteins, making it hard to generate an attack against cancer cells with T-cells that recognize normal self-peptide/MHC. Worse, overcoming this protective response could lead to an auto-immune attack on normal, healthy cells. Therefore, we want to make or use T-cells that recognize mutant-peptide/MHC combos. Such cells would not attack or be effectively suppressed in normal tissues, but could attack tumors.
However, to get these ideal, tumor-targeted T-cells, we need the tumor to produce mutant proteins whose peptide fragments ARE DISTINCT from the peptide fragments of normal proteins. Some tumors have far more of these kinds of mutations than others, and not every mutation is immunogenic (not every mutation can be differentially recognized by T-cells). For example, point mutations (single amino acid changes) that activate proto-oncogenes like BRAF don't necessarily lead to the production of structurally different peptide fragments. A larger mutanome offers a greater chance of immunogenic mutations.


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Human Evolution in Modern Times
I understand that evolution occurred to form the current hominids from a common ancestor millions of years ago. As evolutionary processes take a long time, is there proof of evolution occurring with humans today?

I don't think this necessarily needs to be closed. A good answer would simply point to some recently observed evolutionary trends in humans.

@canadianer i too support you i.e. downvoting a new user at his first post itself would be very bad

Whats wrong with this question? It's short and legitimate, so why downvoting it?

With @Chris editing, there is definitely no reason to not reopen this post.

Common examples of recent human evolution include Lactase (and its associated selective sweep), sickle-cell disease and malaria resistance in zone where the prevalence of malaria changed, height, skin color, blood type and finally various diseases linked with bottleneck. That would be good to have an answer that encompass at least all these examples.

I agree with @Chris's edit. The question is improved.

Yes, there are examples. First: If you want to say, that humans and monkeys evolved, than it is better to say that they evolved from a common ancestor. This makes quite a difference.
If you are looking for examples of human evolution then one of the most obvious traits under evolutionary selection is pigmentation. There is a clear correlation with lattitude (and this UV-index):

The changes in pigmentation occured - seen from a evolutionary perspective - pretty recent when humans moved out of africa and to other regions (see here).
See these papers for more details:

The evolution of human skin coloration
Colloquium paper: human skin pigmentation as an adaptation to UV
radiation.
Epidermal pigmentation in the human lineage is an adaptation to
ultraviolet radiation.

Other examples would be the tolerance for lactose (the sugar contained in the milk) in adults which arose during the last 5-10.000 years. Normally only babies can process these sugar which is contained in large amounts in the mothers milk. When they are weaned, usually the enzyme is not expressed anymore. However with the settling of the humans and the domestication of cattle this changed, so we can still drink milk and eat milk products without producing intestinal problems. See here for more details:

Genetic signatures of strong recent positive selection at the lactase
gene.
Adult-type hypolactasia and regulation of lactase expression.

A third example would be the sickle-cell anemia, which happens due to a point mutation in the human hemoglobin protein. This causes an aggregation of the hemoglobin molecules in the red blood cells which then leads to a reduced elasticity (see here for more details). It also leads to a protection against malaria (most likely by the reduction of the life span of the erythrocytes). The areas with high prevalence of the sickel cell anemia in africa correlate pretty nicely with the distribution of malaria.

An Immune Basis for Malaria Protection by the Sickle Cell Trait
Sickle Cell Anaemia and Malaria

If you look further, you will find a number of different examples, where evolution is present after humans went through a genetic bottleneck (meaning the number of humans was drastically reduced).

Lactase persistance is evidence of recent and ongoing natural selection in humans.
Mammalian young produce lactase, an enzyme needed to break down the lactose sugar in the mother's milk. The young quit producing lactase after they are weaned because they no longer drink milk. Most humans (roughly 70%) also quit producing lactase when they are young. Without lactase, the adults can't break down the lactose sugars if they consume milk. The lactose builds up in the gut, which supports large populations of bacteria that cause gas and indigestion. This is lactose intolerance.
The remaining humans (about 30%) continue to produce lactase into adulthood. They can digest the lactose milk sugar so they do not suffer lactose intolerance. The persistence of the lactase enzyme in adults is associated with the human domestication of cattle.  A study by Todd Bersaglieri and colleagues (2004) showed that a mutation that allowed for continued lactase production spread rapidly by natural selection through populations in north European-derived people between 5000-10,000 years ago.
Interestingly, a separate study by by Sarah Tishkoff and her colleages (2007) identified a different mutation that also allowed for continued lactase production spread quickly by natural selection through populations in east central Africa during the past 7000 years.
Together these studies show how natural selection can act very quickly in humans. Two different mutations occurred in two different populations of humans. Both mutations, however, gave the same selective advantage of lactase persistance in developing agriculturally-based societies.
Literature Cited
Bersaglieri, T. et al. 2004. Genetic signatures of strong recent positive selection at the lactase gene. American Journal of HUman Genetics 74: 1111-1120.
Tishkoff et al. 2007. Convergent adaptation of human lactase persistance in Africa and Europe. Nature Genetics 39: 31-40.


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Can viruses that normally infect eukaryotic cells also infect bacteria?
Can standard viruses infect bacteria?  I'm not speaking of bacteriophage but typical RNA and DNA virus such as influenza and Ebola.

That's a very human-centric view.  There are more bacteriophages floating around this planet than anything else.  Phages are your "typical" viruses, Influenza and Ebola are the weird ones.

All viruses that infect bacteria are known as bacteriophages. In your question you mention the "typical RNA and DNA viruses", however, bacteriophages also contain DNA, which is often double-stranded DNA.

Some viruses even infect other viruses! There are still very few examples and in addition to that those examples involve host species that are on the boundary of the definition of what is a virus. You may want to have a look to mamavirus and its parasite: sputnik. Also one may want to have a look to Marseillevirus (named after the University of Marseille), Lausannevirus (named after the University of Lausanne) and mimivirus.

I'm not so sure about my answer. But I think "bacteriophage" is a polyphyletic name of which was defined by the ability of infecting bacteria, but not defined by the genetic, evolutionary, or morphological relationships.
It seems that if a species (or a taxon in a higher taxonomic level) of virus is infectious to bacteria, it will be classified as a member of the bacteriophage.
Here is a review article about the phage classification.

Tailed phages (Caudovirales) are an ancient monophyletic viral taxon, and include species that infect bacteria and species that infect archaea. People often use the term bacteriophage as a synonym to them. Caudovirales are the most abundant biological entities on the planet. There are three families: Myoviridae, Podoviridae and Siphoviridae, which are characterized by linear dsDNA genomes and unique head-tail morphology. Eukaryote-infecting Herpesviridae are believed to be related to Caudovirales.

The word infect is an encapsulation of several activities: transcription, translation, viral entry, budding, lysis, etc. When you ask if a eukaryotic virus (influenza, ebola) can infect a prokarytoic cell (bacteria) you are asking if these viruses can do the same activities. The answer is no.

Translation is mediated by ribosomes, which are different in
prokaryotes/eukaryotes.
Transcription is mediated by viral RNA polymerase which may be contained in viral nucleic acid. This is possible, but useless without translation of the mRNA.
Viral entry is mediated by cell surface features and would be incompatible for eukaryotic viral entry into prokaytoic cells, and the reverse.
Viral budding is mediated by viral proteins inserted into the cell membrane. This is prohibited since a eukaryotic virus is unable to enter a prokaryotic cell/translate its proteins.
Lysis is mediated by excessive buildup of viral particles inside a cell. This is also prohibited by an incompatibility of eukaryotic/prokaryotic cell surface, translation machinery.


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Fermentation to acetic acid
How much time is needed for the fermentation to acetic acid production cycle, and the conversion of apple vinegar in a sealed container incubated at 37 °C?
Should be in the fully closed?
What is right yeast?
I do it with powder, grated apple in a plastic soda bottle and I add a few peas and a slice of bread. I am waiting for make that.
If you have a better idea on what to do to get a good apple cider vinegar produce in my own house.

A Google search for "homemade apple vinegar" yields lots of information. Perhaps you will get an answer from an expert here, perhaps not.

Ask this on Seasoned Advice, perhaps?

@LittleWhiteLithe Have you a good idea for it?

Ineresting idea. I have once done this in a lab course - and will definitely try the recipe listed below (which is pretty similar to the instructions we used).

There are some recipes available on the web, my answer is based on this webpage and this information:
The process itself is rather easy and you can either use ready bought apple juice, homemade juice or apple cider (basically unfiltered apple juice). You will need some specially cultivated yeast, baker's yeast is generally not recommended since it might produce a lot of unwanted (and probably bad tasting) side products.
For the fermentation itself you need some (very) clean vessels in which the process can take place. These should be from a material which is not corrosive, as the vinegar would otherwise react with the vessel. Food-grade plastic, glass or stainless steel should be ok.
To have the reaction take place, the process need oxygen, otherwise the reaction will stop at the step of ethanol. This is also why acoholic fermentation is always sealed against the air, to avoid the oxidation of the alcohol the acetic acid. A cheese cloth will leave oxygen in, but keep insects and other contaminations out. If you need to filter the vinegar at the end of the process, a standard coffeefilter is good enough.
The following step by step directions come from this website:

Step 1 - Make or buy your apple cider or apple juice
Apple juice or apple cider is the starting point for making apple
cider vinegar. You can make your own, or buy it ready made, fresh,
bottled or frozen.  You can use a mechanical or steam juicer to
produce the juice / cider. To make your own juice (which is the same
as plain unfermented apple cider), see this page. Keep in mind that
you want to select sweet apples, like Fuji, Delicious, Mutsu, Gala,
etc. Green, unripe and unsweet apples (like Granny Smith) do not have
enough sugar to make good cider vinegar. Tips about selecting apples:
Apples used for cider don't have to be flawless. They do, however,
have to be free from spoilage. You can use blemished apples and small
sized apples. You can mix apple varieties together or use all one
variety. The only rule is to cut out any spoilage areas on otherwise
good apples. Spoiled areas will cause the juice to ferment too rapidly
and will ruin the cider. Don't use apples that appear brown, decayed
or moldy. Apples should be firm and ripe. Green, undermature apples
cause a flat flavor when juiced. The best cider comes from a blend of
sweet, tart and aromatic apple varieties. A bushel of apples yields
about 3 gallons of juice.
Step 2 - Make your starter yeast
Adding yeast to activate fermentation is not essential, but will speed
up the process and can produce a higher quality. Special cultivated
yeasts are available for this purpose at wine-making shops and
biological labs but bread yeasts are not recommended. To make a
starter, crumble one cake of yeast into one quart of cider and mix.
This makes enough starter for 5 gallons of cider; double the recipe
proportionately when making more.
Steps 3 - Making Alcohol and Acetic Acid
Pour all of the juice or cider into one or more containers to about
three-quarters capacity; do not close the lids on the containers.
Instead, cover the openings with cheesecloth secured with a rubber
band or string.
Step 4 - Store out of direct light, but some place with constant temperature
Stir the mixtures daily, making sure the cheesecloth is put back in
place. Keep the containers away from direct sunlight and maintain the
temperature at 60 to 80 degrees F.
Step 5 - Keep stirring daily, monitoring and tasting for the next 3 to 4 weeks
Full fermentation will take about 3 to 4 weeks. Near the end of this
period, you should notice a vinegar-like smell. Taste samples daily
until the desired strength is reached.
Step 4 - Filter Mother of vinegar
When the vinegar is fully fermented, filter the liquid through several
layers of fine cheesecloth or filter paper—a coffee filter works well
for this. This removes the mother of vinegar, shown at right,
preventing further fermentation or spoilage of the product. Mother of
vinegar is completely harmless and the surrounding vinegar does not
have to be discarded because of it. It can be filtered out using a
coffee filter, used to start a bottle of vinegar, or simply left in
and ignored.
Step 6 - Done! Storing Your Vinegar
The vinegar is now ready for storage in separate, capped containers.
Stored vinegar will stay in excellent condition almost indefinitely if
it is pasteurized. To pasteurize, heat the vinegar before pouring it
into sterilized bottles, or bottle, then place in a hot water bath. In
both cases, the temperature of the vinegar must reach at least 140
degrees F to pasteurize the product, and should not exceed 160 degrees
F. Use a cooking thermometer to ensure the correct temperature is met.
Cool the containers and store at room temperature out of direct
sunlight.

Thanks Chris. Seems perfectly good answer.I will try this method.

@M007 I missed your initial post, otherwise I would have answered it much earlier.


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Appropriate Buffer for electerophoresis of DNA & Protein TBE or TAE?
Which buffer is best for DNA Electrophoresis and which is best for Protein to be have a sharp bond?
Considering a higher electrical conductivity compared to TAE & TBE and the generation of less heat. If anyone has a particular formulation (add other materials to improve quality) which is obtained as a result of practical experience. I would be grateful if you send me that.

The standard for DNA agarose gel is TAE and for the protein, it depends on the size of the protein and the gel type used! Some times MOPS works best and sometimes Tris-acetate works best. It really depends on the gel used and also the protein and its size. You just have to experiment with them and see which works best for you! This document by Invitrogen might be of help (http://tools.lifetechnologies.com/Content/SFS/ProductNotes/F_050711_NuPage-TS-TL-MKT-HL.pdf). To prevent apparatus over heating, put them in a cold room. I can give you my TAE components if you wish!

@Bez please introduse why TAE stand a standard buffer for DNA (are it works for any size of that even in PFGE?).If you have a table for size of proteins and relative buffer that can complete your upper idea about proteins buffer difference please put complete of them in a answer form.

TAE is the standard DNA agarose gel buffer according to Sambrook et al., 1989 and is used by practically every scientist that I know of for running agarose gels for DNA. Please look at figure 2 for the best gel to use for your protein of interest (http://www.lifetechnologies.com/uk/en/home/life-science/protein-expression-and-analysis/protein-gel-electrophoresis/protein-gels/novex-nupage-sds-page-gels/nupage-sds-page-bis-tris-gel.html). Since the question is on hold I cannot add a new answer but as soon as it opens I will put my comments as a full response.

It would be great if this could be reopened so we can write proper answers and not have to use the comments for this.

The question which buffer for DNA is better is quite old. Both have their pros and cons and I list a few of them:

TBE is a better conductor and is thus less prone for overheating the
gel
Borate is a powerful enzyme inhibitor, so if you want to apply
enzymatic steps downstream, TAE is the better choice
TAE gives a better resolution for large fragments
TBE has a higher resolution for fragments <2kb so it is better here

Both buffers appeared in the 70s when the electrophoresis of nucleic acids was developed (in fact it was derived from protein electrophoresis)
and haven't been changed or optimized much since then, although there is a number of disadvantages:
First EDTA is not really necessary for DNA electrophoresis (it is for RNA, which is also the most likely reason it is still in there). Then
Tris is not the best buffering substance here. Besides being relative expensive it creates a temperature-current feedback loop,
meaning the higher the current gets, the higher the temperature will be. This leads to a higher gel temperature with all the
negative effects of it. For more details on this, have a look into reference 1.
There has been some research on this matter and there are some suggestions available for better running buffers (details are in references 2 and 3).
In short these are:

10mM sodium boric acid (Na2B4O7/Borax): separation of DNA fragments
from 100bp-5kbp
5mM lithium acetate (LiOOCCH3): separation of fragments longer than 3
kbp
1mM lithium boric acid (Li2B4O7): separation of small DNA fragments
and ssDNA

The borate in this buffers is still problematic in terms of a possible enzymatic inhibition, but this can be overcome with a simple ethanol
precipitation of the DNA (which of course also applies for use of TBE buffer).

History and principles of conductive media for standard DNA
electrophoresis.
Sodium boric acid: a Tris-free, cooler conductive medium for DNA
electrophoresis
Ultra-fast high-resolution agarose electrophoresis of DNA and RNA
using low-molarity conductive media

I have had good experience using a lithium boric acid buffer from Faster Better Media. I use it for RNA gels, but it's advertised for DNA gels. I don't think it can do protein, but I've never tried it. I'm not an electrician, but higher conductivity may be the opposite of what you want. The lithium boric acid buffer claims to have less conductivity than a tris based buffer, allowing you turn the voltage up much higher while producing less heat. The higher voltage pushes the DNA down the gel faster, and keeps the bands sharper. My RNA gels take 30 minutes to run, used to take 3 hours. Of course, those slow gels used a MOPS based buffer and formaldehyde to denature the RNA.

The funny thing is one of the founder of Faster Better Media wrote the articles I cited :-)

Grossly, it does not matter what buffer you use. It is the pH that matters.
For DNA electrophoresis EDTA is added in order to chelate divalent cations that serve as cofactors for nucleases. Tris is the base of the buffer and is used to set pH. Along with Tris one can use Boric acid, Acetic acid or phosphoric acid for adjusting the pH.
The buffering range can be obtained by the Henderson-Hasselbalch equation.
The choice of buffers depends on pH range, ease of preparation/storage and fine resolving properties and runtime properties such as heating etc (also mentioned by others).
MOPS is another buffering agent. I am not very sure why MOPS is used instead of Tris for RNA gels; perhaps the latter forms adducts with formaldehyde  by Mannich's reaction whereas MOPS cannot undergo that reaction. MOPS is also a Zwitter-ionic species and maintains neutral pH. RNA should not be kept in alkaline pH. At acidic pH, RNA will become neutral and will not move.
For protein electrophoresis things are a little different. Glycine-Chloride buffer is used because it allows stacking.

There are already many great answers to your question, however I thought I put my comments in form of an answer.
The standard for DNA agarose gel is TAE and for the protein, it depends on the size of the protein and the gel type used! Some times MOPS works best and sometimes Tris-acetate works best. It really depends on the gel used and also the protein and its size. You just have to experiment with them and see which works best for you! This document by Invitrogen might be of help. To prevent apparatus over heating, put them in a cold room. I can give you my TAE components if you wish!
TAE is the standard DNA agarose gel buffer according to Sambrook et al., 1989 and is used by practically every scientist that I know of for running agarose gels for DNA. Please look at figure 2 for the best gel to use for your protein of interest, which has been shown in form of a table.


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Physiological effects of electrical shocks on human body depending on the energy
When discussing safety of electricity, one usually considers a constant DC or AC current with constant amplitude over a longer time. It is easy to find tables in books or in the web which lists different magnitudes of current and it's physiological effect on the human body. For example this (chest pathway):

If you consider a short time discharge, for example from a capacitor or an inductor, this description no longer applies. For example if you charge a small parallel plate capacitor with several thousands of volts and touch it, nothing dangerous will happen (just a slightly painful sensation). Initially the current through the body will be very high (such that it would be deadly if it was constant), but which decays exponentially in a very short time.
So one needs another scheme to evaluate the dangers to the human body in the case of a short discharge. One simple criterion is the energy criterion where one calculates the energy which may maximally deposited in the human body by the discharge. This is for example used in for electrical safety guidelines for German schools (http://publikationen.dguv.de/dguv/pdf/10002/si-8040.pdf, page 27), where for > 60 volts a discharge energy below 350 mJ is considered as safe.
My question is if there exists also an overview of the effects on the human body for short discharges depending on the energy (for the case of > 60 volt), or even a two dimensional map (one axis voltage, the other one energy).
If so, do you have a reference to the original papers.

Is there any source claiming that capacitor discharge with thousands of volts is not fatal?

@JM97: I tested it several times. But be aware, that this only works, if the discharge energy is low enough. Take for example the 350 mJ limit I cited in my post. Below this it should be safe. Then take a parallel plate capacitor without dielectricum, plate distance say 0,5cm and a square plate with $0,25\cdot 0,25 ,\mathrm{m^2}$ area. Then you get a capacity $C = \epsilon_0 \frac{A}{d} \approx 1,1\cdot 10^{-10} ,\mathrm{F}$ and with $U = 500,\mathrm{V}$ you get a  discharge energy of $E = \frac{1}{2} C U^2 \approx 1,38 ,\mathrm{mJ}$.

However take another commercial capacitor with say $1,\mu\mathrm{F}$ capacity, then at $5 \mathrm{kV}$ you get $12 , \mathrm{J}$ as discharge energy. I guess this would be fatal (don't worry, I am not going to test this :-)).

If there is more transfer of charge in less time that is higher amperes of current is passing then it will be fatal not only due to its effects on membrane charges but also due to intense heat produced by the fast moving charges. So the tables given in question are enough


text:
Why doesn't life produce diamond, graphene, nanotubes, etc? Could it?
It seems that life is really good at assembling carbon into extremely complicated molecules (e.g. DNA). Graphene is stronger than any materials currently used by biology. Diamond is harder than any biological materials. Do we know about some inherent difficulty that prevents this? Is there any reason to think that humans will never be able to genetically engineer say, bacteria/fungi/ourselves, to produce diamond, graphene, carbon nanotubes, etc?
I'm looking for specific metabolic hurdles that would make this difficult or impossible. (For example, enzymes/proteins can't do that for this reason, or ATP metabolisms don't have enough energy to form the needed bond.)

well, life, meaning humans, are capable of producing artificial diamonds, i.e. https://en.wikipedia.org/wiki/Synthetic_diamond I would suggest you look into energy required for creating diamonds (3000 deg C, 3.5 GPa) versus energy biology is able to apply in biochemical reactions

Possible duplicate of Why do some bad traits evolve, and good ones don't?

as aaaaaa stated, diamond require high heat/pressure to create. Zoological bodies/tissues can't do that. Also, you should review production techniques for creating graphene and try to imagine how a biological organism could accomplish a similar task

Although your post is interesting, your main question, " Is there any reason to think..." qualifies this question as being primarily opinion-based (i.e., "answers to this question will tend to be almost entirely based on opinions, rather than facts, references, or specific expertise."). I would suggest editing your question to avoid closure.

Biology does not have too many uses for super hard but brittle materials like a diamond. The hardest biological materials are also very tough as well since they need to form complex shapes for maximum utility. Limpet teeth are the hardest biological material known but are also incredibly strong, stronger than steel, not super brittle like diamond1.
As for graphene, the big problem there is reactivity; graphene is a great substance except it is pure carbon and has a huge surface area. If you put it in a solution full of carbon reactive chemicals, it starts doing weird things, and it has particularly weird interactions with cell membranes. Structurally, it is far more trouble than it is worth in something made of cells2.
You also have to remember that, in evolution, it is generally about "good enough", not perfection. Making a pure carbon structure would be an incredibly costly process in a biological (and thus aqueous) environment.
References:

Extreme strength observed in limpet teeth
Exploring the Interface of Graphene and Biology

Good answer, but could I also suggest the problem of breaking it down again. If a biological structure is damaged, or the organism needs to grow, it has to be able to break down its structure - this could be difficult for a substance as stable as graphene or diamond

I think there are plenty of uses for diamonds. Rodent teeth are composed of a hard, brittle face with a softer, tougher backing structure. The hard face could be replaced with diamond.
Grass grows phytoliths which would be a perfect application of even highly brittle diamonds.
Composite materials are usually combinations of hard, brittle materials with high tensile strength fibers (e.g. reinforced concrete, epoxy fiberglass). Diamond reinforced by carbon nanotubes would seem to be the ultimate expression of this strategy.

considering rodents can already chew through concrete with teeth made of a salt composite diamond is not going to gain them a whole lot. and far more costly to make while not necessarily showing better wear resistance.phytoliths are made from silica which is basically free (the plant absorbs some by accident as it tries to dissolve other useful minerals out of soil. daimond would compete for cellulose and sugar for carbon usage.

diamond by their very structure do not bond well with anything, they are all but useless for composites.

@gilleain: Breaking down a structure isn't the only solution.  Many animals (sharks, for instance) shed worn teeth and grow new ones.  Many arthropods shed their carapaces and grow new ones, snakes shed their skin, and (as I am all to well aware of right now) dogs & horses shed their winter coats.

@John If you google for diamond composite there are a lot of hits including at least one commercially available product called Versimax. My chemistry is a little weak, but I don't think it's necessary to form a molecular bond with a matrix material in order to form a useful composite with it. I don't believe epoxy and fiberglass form molecular bonds.

They do bond that would be the reason for epoxy as opposed to just just using a bulk filler. you can make composites with only mechanical interaction but biological composites need to be built on molecular level casting and such are not an option. As for diamond composites note they are all either metals or ceramics, not materials known for occurring in organisms and both requiring absurd temperatures.

Biology is way ahead of us in producing stuff with useful properties. Nanotubes are nothing new, some structures in your body are made out of nanotubes, take e.g. your tooth enamel. The nanotubes there can become depleted of minerals after eating foods containing acids, but they'll refill in about an hour's time. This is why your dentist will tell you to not brush your teeth immediately after eating a meal. Empty nanotubes are easily damaged, they won't be replaced so you'll have permanent mineral loss.
Diamonds crystals have no known use for ling organisms and they are also hard to grow in a biological setting. You have to keep in mind here that humans are used to build structures on a macroscopic scale. Machines that we build will have smallest functional parts that are still enormously large when viewed at the atomic scale. This then means that our machines are prone to degradation due the system accumulating damage at scales that are smaller than the smallest accessible scale.
Biological systems don't have this problem because the relevant machine parts here are at the molecular scale. Damage at smaller scales requires bonds between atoms to be broken which requires very high energies. Also the systems themselves are small enough to intervene at that scale. So, the reason why we want to use diamond (a strong material that doesn't degrade) is not relevant for biological systems.
Here we also need to keep in mind that biological systems are constantly at work to maintain themselves. So, it's not like any machine that we build that only very occasionally will need downtime for maintenance and still we'll take it for  granted that it will degrade over time. A biological system will almost immediately fall apart if the internal self maintenance processes were to stop.  It's the equilibrium between breakdown and rebuilding that keeps your body in shape. Increasing the demand for rebuilding by exercising will shift the balance such that your body becomes stronger.

If hard materials weren't useful for biological organisms, they wouldn't make them. Biological organisms do make hard materials - teeth (as you mentioned), scales, bones, claws, pincers, exoskeletons, bivalve shells, diatom shells, beaks, phytoliths, etc. Diamond is highly resistant to acid, which would prevent cavities. Its hardness would greatly reduce tooth wear in herbivores ingesting phytoliths or digging rodents. Diamond faces on compound eyes would reduce scratching.

@hexagon Yes, I agree here, but do note that in biology the stuff that is used is also being manufactured at the same time. So, you're bones are constantly being broken down and also build up. For diamonds to be used would require the machinery to make the stuff from diamonds to be there at work too. We tend to think of the building phase as being separate from just having that stuff after it's being build, it's then a static thing. To appreciate that biology is different, you should watch these videos https://www.youtube.com/watch?v=SNiiLfB8s0s and https://www.youtube.com/watch?v=zkGb12xBKlM

Bones are created that way, yes, but I don't believe that is the case for all hard materials. Exoskeletons are molted. Snail and bivalve shells are shaped to grow indefinitely. I suspect phytoliths never shrink.


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Anti-antibodies for preventing transplant rejection
Transplanted organs from the donor are attacked by antibodies from the recepient. So can't we construct anti-antibodies and destroy specifically those antibodies which are causing the rejections ? (Instead of giving immuno suppressants)
Well, I can think of some problems :

You would have to administer this for life time which is really expensive.
So, what if anti - antiantibodies were made by the body ?

Any other problems you can think of ?

The root of the problem in this case are not the antibodies but the antibody producing cells (APC). They are capable of producing vast amounts of antibody, so I doubt that this approach would be successful. The problem with targeting these APCs is, that you have to know exactly which are the ones which cause the problem, if you want to target them specifically.

yes, the no. of clones our immune system can produce after it decides on one is enormous. Good point !

Do u think anti-antiantibodies could be made by our body ?

@Biogirl: No. Antibodies against human antibodies would be sorted out in the differentiation process. It would be fairly dangerous to have such antibodies, since antibodies mainly differ between each other in the site which recognizes the antigen. The rest is highly conserved. So it would be very likely to direct the anti-antibody against these parts and then hit all antibodies.

Within an organism, antibodies which bind to other antibodies in the body would be eliminated during clonal selection.  They are not made by the body after that period, which is in the first few months of life.
Such antibodies would have to be introduced from the outside.  Many have been designed or engineered for biotech and as therapeutics.

Problems:
Where are the anti-antibodies created? In a mouse or in a human? Well, of course, you can't use humans to create antibodies so we are left with mice. So what's  wrong with that?

Anti-antibodies will be created to the Fc (the constant section of the antibody) region of the antibody because the antibody is foreign and is treated as a foreign antigen.
So, you would have to use chimeric (or humanized) antibodies... I believe they are pretty expensive and takes a long time to make. Let's see that in current time. By now, I am sure you have heard about ZMapp, the drug that treats Ebola. Well, creating that drug is similar to this because they are creating antibodies as well. But I heard that a few doses of this drug takes 3-6 months to make! In this article , it mentions that it takes half a year to make ZMapp!

In conclusion, it's a real challenge, which is why we haven't found drugs that work like how you describe out in the market... But it's nice that you are thinking about this stuff...

Antibodies are already widely used in treatment of organ transplant rejection. However their targets are not donor specific antibodies as you proposed. Current anti-rejection antibody based treatment targets T lymphocytes (Basiliximab/Anti-thymocyte globulin/Anti-lymphocyte globulin/Campath etc.), B lymphocytes/plasma cells (Rituximab/bortezomib etc.). Here is a slightly old review of some of these antibodies (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2840233/).


text:
Transcription rate expressed in microarray per hour
This article gives measurement of transcription rate  and the unit they're using is microarray per hour.
For example, at 27°C the average expression of their genes is 236.1 microarray per hour (page 4). What does this unit mean? Given that each DNA spot in a microarray contain 1 picomole of DNA (wiki), does it mean that the gene is transcribed into 1 picomole of RNA (or of nucleotides?) per hour? Or does it mean that 236.1 RNA are created every hours?

The units here are relative units of intensity.  There may be about a picomole of probes on a microarray spot, but the units of intensity are not scaled to the precise concentration of DNA on the spot.
There are many variables which make exact measurements of intensity difficult to translate into the number of RNA bound to a spot.  The main one is nonspecific or non-target binding.  The oligomer on the array surface is typically between 25 and 50 bases long - it isn't the entire length of the RNA being tested by a given spot.  Another RNA may have some or most of the same sequence as a probe, or some labelled RNA will tend to get associated with some spots.  Usually factors such as the position of the spot on the array surface, the GC composition of the probe and variations in the batch of RNA preparation will also cause the intensity to vary.
Still, even with all this, with the same prep conditions and with an identical set of arrays, its valid to look at the intensity over time.  In this paper, the investigators took array measurements at different time points and at two different temperatures after 4-Thiouracil  is added to the cells, which allows one to follow RNA produced by transcription at a given time.
Since the units are arbitrary but still proportional to the mRNA found in the cells, a difference experiment like this is valid, even if the absolute concentration of any given RNA is still a bit of a mystery.


text:
what is the principle behind Allele Frequency Net Database
I just wanted to know what is the basic principle of Allele*Frequency Net Database. How are the frequencies present in them calculated??

Allele frequency is simply the frequency at which a specific allele is found in members of a population. Let's take the following example from the site you referenced:

The first column merely has the name of the allele, A*01. The second column is the population from which individuals were genotyped. Skipping ahead, the fifth column is the number of people genotyped from the population, 187 in this instance. Of those 187 people, 14.8% of them have at least one copy of the allele as stated in the third column. It's important to remember that each individual could potentially have two copies of the allele.
The fourth column gives the actual allele frequency, calculated by the number of A*01 alleles/number of total alleles (374, assuming no serious mutations). Of those 374 total alleles, 7.4% (0.074) of them are the A*01 allele, meaning 27-28 of the 187 total people had the allele. Since 14.8% of people had the allele, but the frequency of the allele was only 7.4%, we know that each person who has the allele only has one copy of it.


text:
PCR cycle problem
IF I began a PCR cycle with 5 copies of a particular DNA section, and copied the section by PCR, for 6 cycles, how many copies of the DNA (include the originals) would I have by the end of these cycles? Explain or sketch out your reasoning.
Edit: I think it is 315 copies (5+10+20+40+80+160 = 315) after 6 cycles right?

What do you know about PCR? Especially about the double rates?

I think it is 315 copies after 6 cycles right? I did some research haha

Then you should redo you research. How do you come to 315?

uhh I did 5+10+20+40+80+160= 315

This is exponential - you double the number of molecules in each step. See my answer below.

PCR ideally doubles the number of amplified DNA molecules in each cycle. So after the first step you have 10 molecules, after the second 20, the third 50 and so on. The formula for the calculation is:
n × 2cycles = number of molecules
n is the number of molecules you start your PCR with, cycles is the number of cycles used.
In your case this would be: 5 × 26 = 320 molecules of DNA.

hmmm so it goes 10, 20, then 50? How does it go to 50 for the third cycle?

No. It goes: 10 - 20 - 40 - 80 - 160 - 320. You double the amount of DNA molecules in each step, you are not adding them up.

so in the first cycle the 5 turns to 10? and then you start doubling it up? btw the formula up there I can't seem to find it on the internet, did your teacher teach it?

Yes, since you start with 5 DNA molecules, the first round goes up to 10.


text:
What are the considerations for studying population genetics of bacterial pan-genomes?
I am beginning to work in the field of human gut microbiome, and wondering how (and if) the concepts of population genetics could be applied there.

Considering the competition between the species seems straightforward, although ecological models are probably more appropriate in this case.
Considering the pan-genome of single species however seems next to impossible, as it cannot be divided into independent alleles. One could probably define a number of alleles as the number of copies of a particular gene in the community. However, as it confers fitness on the whole community, it is not clear how to define selection. The notion of genetic drift also seems vague.

I will appreciate clarifications and references to relevant literature.
Update
To clarify the question:

The question is rather theoretical, motivated more by my previous work than by what I am about to do in the field of metagenomics. The latter will be analysis of shotgun sequencing data, where the focus is on identifying new species and comparing the species composition in many patients. I doubt that we will have any data permitting to study the intra-species evolution within a single patient. However, understanding how bacteria evolve to be a particular strain in a particular patient could be a plus: if members of a bacterial community with different genomes live as a group, we can't meaningfully say that some of them are more fit or less fit.
My past experience with population genetics is in the field of HIV, which for most purposes can be treated as a diploid hermaphrodite (each virion carries two copies of the genome, and the genomes are shuffled when a cell is co-infected with several virions). Thus, many basic population genetics concepts are easily applied, although the speed of evolution and the need for constant adaptation add other difficulties (motivating such approaches as fitness edge).
Finally, as was pointed in the comments, the problem is not specific to bacteria - altruism does take place in many communities, and should raise the same kinds of issues as I mentioned previously. I admit that I am not familiar with the necessary theoretical frameworks, and this can be considered a part of the question. I would guess however that most such frameworks do not account for direct gene exchange.

Update 2
A relatively recent article by Eduardo Rocha (2018) discusses the shortcomings of the neutral model (as the principal null model for modern genomics) when applied to procaryotes. This has clarified a lot for me, and I give below a short list of their points:

Mutations in bacteria cannot be treated as neutral.
Polymorphism concept is poorly defined for genomes of varying length, which requires new mathematical framework.
Promiscuity of bacteria, freely recombing and exchanging genes via the horisontal gene transfer makes it difficult to define species and apply many concepts of the traditional population genetics.
Difficulties in separating demographics and genetics (essentially, one needs a combined genetico-ecological approach).

Update 3
Inclusive fitness theory / kin theory (also here) probably fits into this context.

can you clarify why you can't divide the pangenome into different alleles? with present technology the recovery/abundance analysis of metagenome-assembled genomes using parametric (e.g. MetaBAT) or proximity-ligation techniques (e.g. Hi-C) is quite possible, if more intensive than low-pass shotgun sequencing. e.g. https://www.nature.com/articles/s41564-019-0625-0 (full disclosure: i work for a Hi-C company)

@MaximilianPress do you mean that you can assign genomes on a single cell basis? This doesn't make these bacteria independent. E.g., a gene possessed by one of the bacteria may benefit its neighbors of the same species more than itself - how do you define selection in this case? Perhaps, you know a lot more than I about this question, and could expand it in an answer?

I will try to answer the question, but i think i still need to know why you think "Considering the pan-genome of single species however seems next to impossible, as it cannot be divided into independent alleles." I don't really know what you mean by this. If you actually laid out the data you are thinking of using and a couple of details about your proposed methodology, then that would likely help. For example: you are probably sequencing, but with what? Nanopore, Illumina? Are you just doing 16S and OTU counts? How deeply? How are you representing the data as "alleles"?

Also.... when you write "a gene possessed by one of the bacteria may benefit its neighbors of the same species more than itself - how do you define selection in this case?" How is this any different from altruism in any other population genetic context (i.e. usually ignored)? Editing your question to explain some of these implicit assumptions might make it clearer.

There is actually quite a growing field of study examining the community dynamics and "succession" of bacterial communities in people's guts. I'm not sure if this would inform your particular study in the way you're looking for, but it might be interesting to peruse. I'm away from my office right now and can't think of a specific source off the top of my head. If you think it might be useful, I can maybe add an answer that points out some starting points

@theforestecologist Thank you for chiming in. I certainly would appreciate your answer. Moreover, as I have no background in ecology, I could use some direction here.

It is possible to perform gene-level population genetics analyses on microbiome-derived bacterial species.1,2 † However, the biology of bacteria and the nature of metagenomic sequencing data make such analyses difficult and intractable to many of the well-worked tools of population genetics. Here are some of the things you should keep in mind as you build a conceptual framework for understanding the field:
Bacterial genomes are flexible
The term "allele" connotes gene variants, and the underlying assumption of analyses involving alleles is that each (diploid) individual in a population has one or two alleles of a given gene.  When considering a bacterial population, specifically all the cells that constitute a species, some genes will not be present in all cells. For this reason, "allele" is not frequently used in discussions of complex bacterial communities.  For well-studied species like E. coli and K. pneumoniae with many complete strain genomes, plotting the number of genes found across genomes gives a characteristic U-shaped distribution.3  For a given species, this suggests that many genes are present in most strains, and many genes are present in only one or two strains.  The simple thing to do is to constrain your analysis to the gene set that you observe to appear in all ‡ individuals in a population (the core genome, to use a pangenomics term), but this likely excludes genes that are important for niche-specific fitness like virulence factors, antibiotic resistance genes, carbohydrate degradation operons, etc.  Recently, this problem has been addressed computationally by representing pangenomes as weighted graphs with genomic features at nodes 4 and by building composite reference genomes from strain genomes data encoded as reference graphs.3 #
Metagenomes are messy (but getting cleaner!)
Horizontal gene transfer is likely a frequent occurrence in microbiomes,5 confounding analyses that rely on strict inheritance. Since microbiome sequencing is often short-read, the problem of horizontal gene transfer is exacerbated by the difficulty in reliably assembling transferred genes with specific metagenome-assembled genomes.  As noted by Maximillian Press, there are several ways to address this issue. At the bench, proximity ligation sequencing can supplement traditional metagenomic assembly-by-alignment using the ground truth of physical closeness.5,6 Likewise, single-cell isolation combined with multiplexed whole-genome amplification greatly reduces complexity of assembly.7  At the computer, sequence composition (i.e. kmer content) and coverage profiles can be leveraged to associate metagenomic contigs in bins,8,9 which are like pseudo-genomes.§ If you're lucky enough to have long-read sequencing data, many of the problems associated with assembly of short reads are no longer an issue,10 with the added bonus that SMRT and nanopore sequencing produce methylation data -- unique methylation profiles can be used to infer which contigs likely belong to the same genome or to associate extragenomic contigs (e.g. plasmids) with their hosts.11,12  All of this to say that getting complete genomes from microbiome sequencing is non-trivial, but there are many techniques and tools at your disposal.
Microbiome data is relative
Much of population genetics deals in counted data -- $n$ individuals with allele $a$, $m$ individuals with allele $b$, and so on. Concerning microbiomes, the absolute abundances of individual microbes are not recoverable from sequencing alone. Microbiome data is therefore said to be compositional.  Like RNA-seq, gene or organism abundances derived from metagenomic sequencing are relative, and, importantly, many of the assumptions underlying the statistical analyses applied to absolute counts do not hold for compositional data.13,14  Thankfully, statisical tools for population-level analysis of microbiomes have been developed,15,16 though their widespread adoption has been slow.
Microbiome members are often interdependent
As hinted at in the question update, bacteria display a type of community altruism, where an individual cell with a specific gene can influence the fitness of neighboring cells that lack the gene.  For an example, see my answer to Will all bacteria become resistant against all antibiotics in the long term? concerning secreted β-lactamases.
Therefore, spatial association is an added factor when considering population dynamics of a genetically heterogeneous bacterial species.  Some methods that address cell-cell spatial proximity in microbiomes include sequencing of cryofractured fragments 17 and probe-based spectral imaging.18 Even if microbes are not spatially associated, different microbes may play complementary roles in the iterative metabolism of large carbohydrates into small metabolites.19,20
Surely, this discussion is incomplete, though I hope my answer has given you the footing you need to continue your own exploration to find the appropriate resources for your research.  For a more in-depth discussion of the points I've addressed here, see What Is Metagenomics Teaching Us, and What Is Missed?,21 particularly the sections titled Strain-Level Analyses and Ecoevolutionary Modeling.

† I recommend keeping up with the publications of Ran Blekhman, Peer Bork, and Katie Pollard.
‡ Core genome membership seldom requires that 100% of individuals represented by that genome contain the gene in question, and setting such a strict cutoff would likely exclude truly essential genes that were not found associated with a given strain due to gaps.  In practice, core genome cutoff thresholds are on the order of 90%.
# The senior author for reference 3 made a great explanatory Twitter thread.
§ Contigs and Bins and MAGs, oh my! As with any field, metagenomics has field-specific jargon. Coursera seems to have a good lecture that includes these concepts and how they relate to eachother.

References

Garud NR, Pollard KS. Population Genetics in the Human Microbiome. Trends Genet. 2020 Jan;36(1):53-67. doi: 10.1016/j.tig.2019.10.010.
Priya S, Blekhman R. Population dynamics of the human gut microbiome: change is the only constant. Genome Biol. 2019 Jul 31;20(1):150.
Colquhoun RM et al. Nucleotide-resolution bacterial pan-genomics with reference graphs. bioRxiv 2020.11.12.380378.
Gautreau G et al. PPanGGOLiN: Depicting microbial diversity via a partitioned pangenome graph. PLoS Comput Biol. 2020;16(3):e1007732.
Kent AG et al. Widespread transfer of mobile antibiotic resistance genes within individual gut microbiomes revealed through bacterial Hi-C. Nat Commun. 2020 Sep 1;11(1):4379.
Stalder T et al. Linking the resistome and plasmidome to the microbiome. ISME J. 2019 Oct;13(10):2437-2446.
Chijiiwa R et al. Single-cell genomics of uncultured bacteria reveals dietary fiber responders in the mouse gut microbiota. Microbiome 8, 5 (2020).
Alneberg J et al. Binning metagenomic contigs by coverage and composition. Nat Methods. 2014 Nov;11(11):1144-6.
Kang DD et al. MetaBAT, an efficient tool for accurately reconstructing single genomes from complex microbial communities. PeerJ. 2015 Aug 27;3:e1165.
Kolmogorov M et al. metaFlye: scalable long-read metagenome assembly using repeat graphs. Nat Methods 17, 1103–1110 (2020).
Beaulaurier J et al. Metagenomic binning and association of plasmids with bacterial host genomes using DNA methylation. Nat Biotechnol. 2018;36(1):61-69.
Tourancheau A et al. Discovering and exploiting multiple types of DNA methylation from individual bacteria and microbiome using nanopore sequencing. bioRxiv. 2020.02.18.954636.
Tsilimigras MC and Fodor AA. Compositional data analysis of the microbiome: fundamentals, tools, and challenges. Ann Epidemiol. 2016 May;26(5):330-5.
Gloor GB et al. Microbiome Datasets Are Compositional: And This Is Not Optional. Front Microbiol. 2017 Nov 15;8:2224.
Shi P et al. Regression analysis for microbiome compositional data. Ann. Appl. Stat. 10 (2016), no. 2, 1019--1040.
Kurtz ZD et al. Sparse and compositionally robust inference of microbial ecological networks. PLoS Comput Biol. 2015 May 7;11(5):e1004226.
Sheth RU et al. Spatial metagenomic characterization of microbial biogeography in the gut. Nat Biotechnol. 2019 Aug;37(8):877-883.
Shi H et al. Highly multiplexed spatial mapping of microbial communities. Nature. 2020 Dec.
Kundu P et al. Species-wide Metabolic Interaction Network for Understanding Natural Lignocellulose Digestion in Termite Gut Microbiota. Sci Rep 9, 16329 (2019).
Selber-Hnatiw S et al. Metabolic networks of the human gut microbiota. Microbiology. 2020 Feb;166(2):96-119.
New F, Brito IL. What Is Metagenomics Teaching Us, and What Is Missed? Annual Review of Microbiology. 2020 74:1, 117-135.

Thank you for clearly restating the points that were somewhat confused in my question. I appreciate very much the reference list - it would have taken me hours of work to pin down all of these (I had seen only Garud&Pollard article).

This is a great reference list.

I will answer this as best as I am able, the updates are helpful for providing context. I'll also note that there are several questions packed in here, which makes it a bit more laborious to answer. I am going to try to answer the following questions:

How can I infer selection on microbiota within single hosts (patients)?
How can I resolve genomes of related organisms from a complex mixture?
How can I do population genetics in the presence of horizontal gene transfer (HGT)?

Question 1
I think that there are some assumptions here that rest on methodological tractability that are, as far as I can tell, not accurate. For example: "...if members of a bacterial community with different genomes live as a group, we can't meaningfully say that some of them are more fit or less fit."
This seems to be the crux of the issue. I would argue that if you are willing to take time-courses from the same patient, it is not at all difficult to measure changes in abundance. Causal inference of e.g. selection is a somewhat harder problem, but one that has been treated exhaustively elsewhere.
If you are willing to postulate that you can deconvolute lineages from a single sample, and measure multiple samples from the same patient, then you can try to measure selection over time. Some steps of this are hard (e.g. if lineages are closely related), but those are merely technical problems that can be solved.
There are also methods that try to infer bacterial growth rates from a single sample, but they're a bit more complicated.
Question 2
Resolution of complex mixed populations into genomes is a well-studied problem. THere are a number of approaches to do this (all of which require relatively high investment and sequence assembly probably):

Long-read single-molecule sequencing: simply use Nanopore or high-coverage PacBio reads to directly ascertain the genome. Expensive (especially for complex samples) but perfectly possible. If you use Nanopore you may not even need to assemble!
Assembly binning: Statistical approaches such as MetaBAT2 attempt to deconvolute metagenomic assemblies based on sequence composition and contig coverage. Limited by the resolution of your assembly- if a contig is collapsed in the assembly, you can't put it in two places (as in the case of very closely related genomes).
Proximity-ligation binning: similar to assembly binning, but using complementary single-cell information to place contigs into the same bin. Some additional refs. More accurate and higher-resolution, but involves an additional data type/expense. Full disclosure: I work for a company that provides this as a service and sells kits to do it.

All of these methods, in principle, could be applied to a subset of samples (e.g. beginning and end) and reference genomes could be imputed to the other samples, where you can just measure them by low-coverage shotgun.
Question 3
This is a big one, I would suggest simply googling it to learn more than I could hope to tell you about population genetic theory in the presence of HGT.
The shorter version is that detectable HGT is not super common at the time scales that you are likely to be dealing with. Most HGT ends quickly with the transferred DNA getting digested for energy or selected out as genomic junk.
Nonetheless there is a clear case that selection of lineages can be driven by HGT events, especially under very strong challenges like antibiotics.
I would argue that if you can measure abundance of a lineage and assign HGTs to genomes in specific samples (readily possible with proximity ligation at least), there is no barrier to doing population genetics and measuring selection. It's just another kind of mutation that happens to have a high rate and to occur in parallel. People measure the selective advantage of e.g. antibiotic resistance all the time.
You may be using heuristics and ad hoc methods for some steps, but genomics is just heuristics and ad hoc methods anyways.

Thank you for this answer - I will certainly return to it more than once, as I immerse myself deeper into the field. Your insights about our ability to access single genomes are particularly valuable. I am not sure why we are locked in a particular sequencing approach (I have recently joined the team) - I will raise the question with my colleagues, when I have a chance.

@Vadim glad that it is helpful. As for the approach, my expectation is that it is 1) cost limited (plain low coverage Illumina is quite cheap these days, the other techs are likely at least $500 sample, minimum), 2) familiarity with the approaches. Bringing expensive new technologies into the lab can be a hard sell when you have infrastructure in place for a cheap approach that is perfectly good for answering most questions. For example, low-coverage Illumina is probably just fine for the  interspecific measurements!