text:
Protein modification and ATP consumption
According to wikipedia there exists a lot of ways to modify a protein (post-translationally). Just to mention few: phosphorylation, dephosphorylation, glycosylation... While phosphorylation requires an ATP molecule, the reverse reaction, dephosphorylation, does not.
How about the other process?
Do all modification respect this scheme (i.e. one transformation consumes ATP but the reverse transformation does not)?
If not, I am particularly interested to know if one transformation and its reverse transformation both requires ATP.
Thank you

ATP is adenosine triphosphate. The reason protein phosphorylation usually consumes ATP is because the ATP contains the donor phosphate group. Some other post-translational modifications have ATP-dependent steps (e.g. ubiquitination), but not most (e.g. acetylation). As you say, there are a lot of ways to modify a protein post-translationally, so why not look at the wikipedia pages for the various modifications to see which ones have steps that require ATP?

I don't think there are any which have direct ATP involvement in both steps. Indirect dependence may be there.


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What makes E. coli yellowish?
LB, TB media, and yeast extract are also yellow but at the heart of the question. What chemical(s) make E. coli yellow?

If I had to guess, I would say FMN/FAD.

I think of E. coli being more white than yellow.  When you compare E. coli and S. aureus on an agar plate, the Staph colonies have a much more distinctive yellow tint.  The yellow tint in Staph colonies is due to staphyloxanthin, a carotenoid pigment.

You will be surprised that when describing the colonial morphology of E. coli on a Blood agar plate, the word choice for colour is "grey". I believe that user leonardo up there had a very good explanation about this "yellowish colour" question.

Bobthejoe's comment is the best answer so far.  Despite many other types of bacterial colonies being "more" yellow than E. coli, E. coli is definitely not white.
Flavins, especially riboflavins, are the predominant compound responsible for this coloration.

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What are good practices with reusing desalting columns
At least according to a few sources Prozyme and Protocols-Online, it is possible to reuse desalting columns and since I'm cheap I would like to also.
Key things seem to be washing with several column volumes of your buffer of choice. Also certain columns like crosslinked desalting columns are not amenable to regeneration. I'm curious about other factors.
I'm mainly using Bio-Rad micro-spins

Regardless of what protocol you use, and what the advertised efficacy of that protocol might be, in any situation like this I think the important thing to consider is: what would happen if the material taken from a re-used column was contaminated by a previous application? Can you live with the consequences of such contamination?
If you are preparing DNA for further use (PCR and/or cloning and/or transformation) then you run the risk of propagating a contaminant through subsequent steps and getting into a real mess. I worked in a lab once where one postgrad ended up spending several weeks working with a cloned fragment that was actually derived from someone else's work in the same lab (although not due to re-use of a column as far as I remember).
If you are preparing protein samples then the risks are possibly reduced, but if the protein sample is going to be subjected to sensitive methods (blotting, MS) then again, could get messy.

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What causes Acne?
What exactly is going on biologically at the molecular level with acne and causes breakouts? Hormonally, there is a lot going on but I'm curious about what is going on chemically.

What are you looking for beyond wikipedia? That seems to cover your questions.

Acne is caused by blockage of hair follicles by oils (sebum) from your skin and dead skin cells. Hormonal changes in puberty cause your skin glands to produce a lot of oil.
Blocked follicles are visible as a small black dot (blackheads).
The recognizable pimples occurring during normal puberty acne form because of bacterial infection (the sebum/skin mix forms a good growth medium for bacteria). The immune system responds with inflammation (the red "base"). The pus (the white "middle") is a dead bacteria, skin and white blood cell mix.
Most treatments for acne focus on either killing the bacteria or removing the dead skin or sebum. Professional medical treatments might also focus on altering hormonal levels. This is also why the COCP , the birth control pill, usually lessens acne in women. Popular over-the-counter remedies for acne (e.g. Clearasil) usually contain a lot of alcohol which both kills the bacteria and removes the sebum, but also makes the skin dry. This in turn causes the skin to produce more sebum thus making the problem worse.

Good answer but it could do with some citations and expansion of "COCP" abbreviation.

And you could've explanded the COCP in your comment @GriffinEvo so that I don't have to google that it means Combined Oral Contraceptive Pill - birth-control pill.

@TomášZato I didn't look it up - I was asking the OP to do it so the correct meaning of COCP was used (if there is more than one). The responsibility lies with the OP, not me, I was just asking for clarification on the abbreviation and other points to make the post meet the guidelines.

You know, you you could've just edited the answer... I added an explanation

I don't like to perform edits that change contents of the answer. I think editing other's posts should merely to improve grammar, formatting and general readability. However this attitude can individually differ.

The anti-acne activity of the combined oral contraceptive is dependent on the type of pill taken. Some pills actually have the opposite effect of worsening acne. This is depending on the type of progesterone in the pill.


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Resuspending Cells from a filter plate
My experiment involves pulling down cells on to a filter plate for a variety of assays. However, I want to ensure that my cells are alive so I am trying to resuspend them so that I can do an accurate cell count. However, I am unable to close off the mass-balance which suggests that the cells are either getting through the membrane or getting stuck.
Any suggestions on the appropriate way to fully resuspend cells? I'm currently using a P200 to mix the solution.

Have you tried trypsinizing them? Depending on whether they're suspension or adherent, and the time they've been on the membrane, you might be able to detach them just like you would from a culture dish/flask. Also, are you using a vacuum to adhere them to the membrane, or just allowing them to settle? Too much pressure may be getting them caught in the pores, although theoretically the pores should be much too small for them to fit through...

@Mattdmo, these are suspension cells so trypsinization will kill them. I'm using a vacuum manifold. I'm fearing the same thing which is why I'm doing this experiment to see if I can complete th mass balance

Resuspending is hard due to attachment/entrapment in the filter and liquid retention around the edges.  Consider using optical live/dead staining on the membrane?   If you are concerned about pass-though you can also try and capture the fluid that comes through the membrane and give it a hard spin in a centrifuge.  Look for a tiny cell pellet on the outside bottom (mark the caps).  Or a simple and cheap dye for viability or counting that would be great on a filter


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Identification of a small mammal
Could someone help with identification of this small mammal? Is it a possum? A dormouse?
It was photographed in Sukhumi botanical garden, in Abkhazia (west of Georgia, on the shores of the black sea).

This is not a marsupial such as an opossum, since no marsupials are native to the region. It looks to be a rodent of some kind, probably a species in the Muridae family.
Which species is it is exactly is hard to tell, since there are many species in Muridae which look quite similar, but my best guess is that this is a Tamarisk jird. It could also conceivably be an escaped domestic gerbil or some type of mouse. The one that you found has more pronounced light areas on its coat than the photographs I found, but is within reasonable limits of what variation can be expected.
Here are some of the few pictures I was able to find that I could verify were of this species.

Klaus Rudloff (source)

Klaus Rudloff (source)


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Pyruvate dehydrogenase: Apparently anomalous NAD/FAD redox reaction
Below is the mechanism for the reactions of the pyruvate dehydrogen complex, which oxidatively decarboxylates pyruvate and transfers the acetyl group to coenyzme A for further metabolism in the Krebs cycle.

The key point is that after $\ce{FAD}$ has reoxidized the dihydrolipoamide, the resulting  $\ce{FADH2}$  is reoxidized by $\ce{NAD+}$, which is reduced to $\ce{NADH}$.
I find the chemical energetics of this difficult to understand. According to standard tables of redox-potential for half reactions, $\ce{FAD}$ is a stronger oxidising agent, and a weaker reducing agent, than $\ce{NAD+}$, which is why it used in the succinate dehydrogenase reaction, for example. How, then, can it reduce $\ce{NAD+}$?
If $\ce{FADH2}$ is really able to reduce $\ce{NAD+}$ it should generate as much ATP in oxidative phosphorylation after oxidation of succinate to fumarate, despite the relatively low free energy change for that reaction. But that is absurd.

This is the most perceptive question on a biochemical topic that I have seen in the last twelve months, and deserves far more votes than it has received. A student, apparently new to biochemistry, has brought his chemical knowledge to bear and pointed to an apparent anomaly which most text-books ignore. The beauty of this question to me is that the explanation for this apparent anomaly illustrates the application of chemistry in a biological context. I have learnt from finding the answer to the question, and I hope the poster's interest in biological chemistry has been stimulated by it.

[1-2] IMO, and as I pointed out (politely) in the comments to the accepted answer, there is no thermodynamic problem here whatsoever. Why shouldn't FADH2 reduce NAD(+)? The reaction is reversible. It may be inferred from the standard redox potentials that the position of equilibrium greatly favours FAD reduction by NADH, but this does not mean that the reaction cannot (thermodynamically) proceed in the opposite direction. All that is required is a negative delta G. The position of equilibrium in ethanol dehydrogenase reaction,for example, greatly favours acetaldehyde reduction, and NADH ...

[2-2] can reduce acetaldehyde (to produce ethanol, as happens in yeast), BUT NAD(+) can oxidize ethanol (to give acetaldehyde, as happens in liver). When dealing with redox potentials and Gibbs free energy, one needs to sharply distinguish between standard conditions and actual conditions. For a reaction to proceed spontaneously (only) requires a negative delta G (or a positive redox potential), not a negative standard delta G (or a positive standard redox potential)

IMO a great reference to this problem is Calculating apparent equilibrium constants of enzyme-catalyzed reactions at pH 7 by RA Alberty. The pdf is freely available

Summary
The redox potential widely quoted in text books for the half-reaction reduction of FAD to FADH2 is indeed inconsistent with the observed direction of several reactions involving this coenzyme, including its reduction of NAD+ in the dihydrolipoamide dehydrogenase reaction. The reason for this is that the value quoted is determined for free FAD in aqueous solution, which does not equate to the situation within the enzyme. In its tightly bound state in flavoproteins, the immediate environment (and, in some cases, covalent linkage) alters the affinity of the flavin ring for electrons, hence causing the redox potential to deviate from that in solution. The extent and direcction of this deviation differs in different flavoproteins.
Restating the problem
The basic idea of chemical theromodynamics is that reactions are energetic favourable if the (Gibbs) free energy change is negative (see e.g. Section 14.1.1 of Berg et al.). Although the actual free energy change, ΔG, is influenced by the relative concentration of the reactants, the usual starting point for assessing this in biochemistry is the ‘standard’ free energy change at pH7 and 25˚C — ΔG0′. In the case of oxidation/reduction reactions the standard oxidation–reduction potentials E0′ of each of the two half reactions are usually known (as they can be measured in solution), and one calculates the ΔG0′ from the oxidation–reduction potentials of the two half reactions, using the formula:

ΔG0′ = –nFΔE0′

What is important for this discussion is not so much calculation of actual values, but determining their sign, as for a negative ΔG (reaction energetically favourable) a positive ΔE is required.
Let us consider an example using the much abbreviated version of Table 18.1 of Berg et al. Biochemistry, below:
Oxidant     Reductant       n     E´o (V)
Succinate   α-Ketoglutarate 2   – 0.67
NAD+        NADH            2   – 0.32
FAD         FADH2           2   – 0.22
Fumarate    Succinate       2   – 0.03

Consider one of the energy-generating oxidation reactions of the Krebs tricarboxylic acid cycle:
α-ketoglutarate + NAD+ → succinate + CO2 + NADH
This is made up of two half reactions:
(+ 0.67 V) α-ketoglutarate  → succinate
(– 0.32 V) NAD+ → NADH
which gives an overall ΔE0′ of  + 0.35 V, i.e. a negative ΔG0′, which is consistent with the actual direction of the reaction and the production of energy. (Actual worked numeric examples can be found in Berg et al.)
Now if we consider the dihydrolipoamide dehydrogenase reaction
NAD+ + FADH2 → NADH + FAD
The two half reactions are:
(+ 0.22 V) FADH2 → FAD
(– 0.32 V) NAD+ → NADH
This gives an overall ΔE0′ of – 0.1V, i.e. a positive ΔG0′, which, as the poster astutely points out, is not consistent with the direction of the reaction.
Resolving the apparent anomaly
Berg et al. do, actually, remark on this in section 17.1.1 on the pyruvate dehydrogenase complex:

This electron transfer to FAD is unusual, because the common role for
FAD is to receive electrons from NADH. The electron transfer potential
of FAD is altered by its association with the enzyme and enables it to
transfer electrons to NAD+.

So, how is “the electron transfer potential of FAD altered by its association with the enzyme”? The quotation from Ghia and Massey1 provides a general summary:

The pyrimidine nucleus of the three-membered ring system is electron-deficient and can be viewed as an ‘electron sink’. In thermodynamic terms, any interaction which tends to lower its electron or negative charge density will increase the redox potential. The reduced flavin consists roughly of an electron-rich phenylenediamine moiety fused with a (4,5-diamino)-uracil. The latter is the moiety in which the negative charge (i.e. two electrons taken up by the oxidized flavin) is localized and, most importantly, stabilized. The degree to which this negative charge is stabilized or destabilized is an important factor governing the redox potential. Thus while a positive charge in the protein around the pyrimidine ring will contribute to increase the redox potential, the presence of negative charge or of a hydrophobic environment will lower it.

In the specific case under consideration — dihydrolipoamide dehydrogenase — it has been established2 that a positively charged lysine-57 sidechain (numbering for the A. Vinelandi enzyme) that increases the redox potential (with, perhaps, a small contribution from the backbone N of alanine-326):

[FAD environment in dihydrolipoamide dehydrogenase from A. Vinelandi. NAD is shown in light blue, except for the O and N atoms at the end of the flavin ring interacting with the protein. A section of the latter is shown as a wireframe in salmon pink without sidechains, except for lys57 and ala326. (Own diagram from 3LAD.pdb)]
A second apparent anomaly and its resolution
In drawing attention to the apparent anomaly in the reduction of NAD+ by FADH2, the poster suggested that this would imply one should by able to obtain enough energy to produce 3ATPs from reducing FADH2. The problem with this is that one actually needs FAD to have a lower redox potential to perform the reduction of fumarate that occurs in the Krebs cycle:

This can be seen by considering the redox potentials in the table above:
succinate + FAD + → fumarate + FADH2
The two half reactions are
(–0.22 V) FAD → FADH2
(+0.03 V) succinate → fumarate
Thus, there is an overall ΔE0′ of – 0.19V, i.e. a positive ΔG0′ — another anomaly!
Berg et al. are rather coy on this one. They do not comment, but in their Table 17.2 of free energy changes in the steps of the TCA cycle — where all the other values are given as exact figures — they quote  “~0” for this step.
This implies a higher (more positive) redox potential for FAD than the –0.22 quoted in the table — certainly not the lower one of dihydrolipoamide dehydrogenase.
I turns out that this is, in fact, the case. In succinate dehydrogenase (aka as fumarate reductase) FAD is covalently attached to a histidine residue (his44 in E. coli) at the other side (position 8) of the flavin ring3.

[FAD environment in succinate dehydrogenase from E. coli. NAD is represented as previously. A section of the latter is shown as a wireframe in salmon pink without sidechains, except for his44 and a backbone N. (Own diagram from 1L0V.pdb)]
An extensive review4 of the redox potentials of covalently and non-covalently bound flavoproteins shows that covalent attachment increases the redox potential.
Final observations

Chemistry is a powerful tool for understanding biology at the
molecular level. Biology is subject to the laws of chemistry, but the
chemistry of the special environments encountered (protein interiors,
spaces compartmented by membranes) need taking into consideration,
something that the classically trained chemist, new to the subject,
may not fully realized.
Elementary textbooks often have to walk a fine line between
comprehensibility and truth. The student of biological chemistry
should realize that the initial simplifications that enabled him to
get an overall grasp may mask greater complexities. One of the
intellectual satisfactions in science can be found by resolving these
complexities in a rationale manner.

References
1Ghisla and Massey (1989) Mechanisms of flavoprotein-catalysed reactions
2Maeda-Yorita et al. Biochemistry (1994) 33, 6213–6220
3Kim and Winge (2013) BBA 1827, 627–636
4Heuts et al. (2009) FEBS Journal 276, 3405–3427

Your answer is gold! I really enjoyed it. The concept you used is called symphoria or Neighbouring group participation or anchimeric assistance in organic chemistry texts. I was familiar with this concept in the field of organic chemistry and how it was used to determine the mechanism of reactions But I'd never seen the actual use of this in biochemical studies. Great job!

(+1) [1-3] Although I don't like criticizing such an excellent answer, I think the following point is relevant: to the sentence "... which, as the poster astutely points out, is not consistent with the direction of the reaction." must be added the qualification "when the reactants are in the standard state". But what determines spontaneity is the actual reduction potential (or Gibbs free energy), not the standard reduction potential (unless standard conditions happen to apply). ...

[2-3]  That is, the conditions within the active site may well favour oxidation FADH(2)  by NAD(+) without the need to postulate any change in oxidation/reduction potential of cofactor by binding to the enzyme. To take an example given by Berg: the LDH reaction.  When reactants are in the standard states (1M), the reaction greatly favours pyruvate reduction. But in the Cori cycle, depending on in vivo conditions, the enzyme may act either as a pyruvate reductase OR as a lactate dehydrogenase.

[3-3] If we were to consider only standard reduction potentials we would conclude that the enzyme always acts a pyruvate reductase.  Similarly, although the point about isocitrate dehydrogenase is a good one, but MDH is surely a counter-example? The position of equilibrium of the MDH reaction greatly favours oxaloacetate reduction (when reactants are in the standard state, oxaloacetate reduction is the 'spontaneous' reaction), but in the TCA cycle MDH functions as a malate dehydogenase, contrary to the above analysis.

@Sam Thank you for your kind words. This sort of effect is fundamental to other aspects of protein chemistry, especially catalysis, where the environment of an amino acid residue can alter its properties.

@user338907 — I agree that an unfavourable ΔGo can be 'overcome' by, e.g. the removal of products producing a favourable ΔG. In a metabolic pathways, this may be especially relevant. In approaching this question I was aware of this possibility. However this does not seem to be the case for dihydrolipoamide dehydrogenase, as there is no "pull" on the NADH produced. I personally was surprised at the extensive literature on the redox properties of FAD, and somewhat ashamed at my ignorance. For me the difference between the diffusible NAD and tightly-bound FAD has acquired another dimension.

In the example of dihydrolipoamide dehydrogenase from A. vinelandi, the interaction of the FADH2 with the protein should ideally decrease the reduction potential, (not increase it). If the reduction potential is decreased, then the protein-bound FADH2 will be better able to donate e- to NAD+. Thus, it doesn't make sense to position positively charged aa residues next to the FADH2 in this particular example provided from A. vinelandi.

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Why is fructolysis independent of glycolysis?
In glycolysis, glucose is converted to fructose-6-phosphate before further breakdown. However fructose, instead of being phosphoryated to fructose-6-phosphate by glucokinase (and thereafter to pyruvate via glycolysis), yields fructose-1-phosphate in the reaction catalysed by fructokinase.
Why is this so?
It should be just as easy to phosphorylate fructose to both compounds. Wouldn’t it be more economical to use a single reaction, so everything would be easier to regulate? As there are several disorders that arise from the deficiency of enzymes used in fructolysis, wouldn’t there be a selection pressure against two separate metabolic pathways?
Edit:
Thanks for David's criticism; I see the confusion, I actually meant that it is mainly metabolized in the liver. I don't really have any real sources as I do not have access to much and quite a large portion of online resources is behind paywall. After a bit of digging I was able to find an overview that reiterates most of my claims. Sorry for not being able to provide more.

In the liver, fructose is rapidly converted to
fructose 1-phosphate via fructokinase. Fructose 1-phosphate is then converted into the trioses dihydroxyacetone
phosphate and glyceraldehyde via aldolase B.

The liver can extract 40–70% of dietary fructose in
both rats and humans. The high efficiency of
fructose extraction by the liver is due to the fact that the
liver is the main source of GLUT-5. Anatomically,
the liver is the first organ to receive absorbed nutrients
via the portal vein; in that way, it contributes to fructose
extraction. In contrast, the liver accounts for a smaller
percentage of glucose extraction for glycogen restoration

Above are two excerpts from Chapter 19 - Fructose-Induced Hypertriglyceridemia: A Review. This source also (albeit vaguely) states that the resulting triose phosphates (glyceraldehyde and dihydroxyacetone phosphate) can and will enter pathways like glyconeogenesis, gluconeogenesis etc.

Up until this point in fructose metabolism, there have been no rate-limiting steps and, as a result, there is an increased amount of substrate leading to metabolic pathways from triose phosphate (i.e. glycolysis, glycogenesis, glyconeogenesis, lipogenesis, and fatty acid esterification).

Another source for my assertion that fructose is mainly metabolized to fructose-1-phosphate (Elsevier's Integrated Biochemistry):

Unique Characteristics—Aldolase B Specificity
Fructokinase, like glucokinase, is found primarily in the liver. Unlike hexokinase and glucokinase, it phosphorylates the sugar at the C-1 position. Aldolase B, which is specific to the liver, works on both F1,6-BP and F1P. In extrahepatic tissues such as muscle or adipose tissue, fructose is phosphorylated to F6P by hexokinase.

Also, a possible lead, from Fructose: Absorption and Metabolism. Encyclopedia of Human Nutrition:

The allosteric inhibition of PFK effectively reduces the rate of glycolysis and decreases hepatic glucose uptake overall. In contrast, the entry of fructose carbons through the pathway proceeds without this limitation.

Using different enzymes (and therefore a different pathway) allows regulation of glycolysis in the liver without unnecessarily affecting fructolysis.

I have edited your question to make it more readable. You need to learn to keep to the point. Written communication requires you to work to decrease the number of words to those that are necessary and then make sure you use the right words. Irrelevant asides inhibit comprehension and the reader will become confused or give up. This is meant as constructive criticism. I shall try to answer your question in due course. Are you sure you meant to write "Fructose is first converted to glucose phosphate before glycolysis"? Do you mean glycogenesis? And which glucose phosphate? Please edit.

And while you are editing your question please give the sources of your assertions, particularly that fructose is not phosphorylated to F 6-P (and do you mean not or only a small proportion is?) and that fructose is is only metabolized in the liver. My text discusses the metabolism of fructose in adipose tissue. Let's try to construct a good question to justify a good answer.

@David Thanks for your helpful edit! Granted, that question was written in a panic and I didn't really proofread it, so it just read like a continuous stream of thought. I have tried my best to add relevant sources; most of my assertions are usually taken for granted, so I didn't think of doing it initially. Glad I did, though! Helped to clear some of the confusion behind this monster of a topic. Also, sorry if the sources aren't up to par, I really can't find anything better.

Thanks for updating your question.  I'll try to find time to research an answer over the weekend, although if someone gets in before me, all well and good.

"Also, why is fructose mainly metabolized in the liver while glucose generally is not?" There are newer evidence suggesting that most of ingested fructose is converted into glucose by intestine cells. If ingested in small amounts it probably never reaches the liver in fructose form. https://www.cell.com/cell-metabolism/fulltext/S1550-4131(17)30729-5

@paymanz The paper you cite seems solid, but if correct it is difficult to see why the liver expresses the enzymes for fructose metabolism. Something doesn't seem quite right. (And the poster's statement that glucose is mainly not metabolized in the liver is not quite right either, or at least misleading, because the liver is the main controller of glucose distribution.) The other thing I don't like about the paper is that is concerned with what happens in fructose excess — i.e.  when there is excess intake of sucrose. That is unlikely to be what the system evloved to cope with.

I have removed the second question about the preference for glucose or fructose by the liver as it is quite separate and should be posed separately. I have also separated your edit from the actual question. It is important people can see what your actual question is — what is the rationale for fructose to be metabolized via F 1-P. Make it clear, but keep it short. I have been busy doing other things (the sun disappeared long ago) but finally have written a long answer, which does not answer the question, but discusses possibilities and ways of approaching such questions.

Summary

The mechanistic reason why fructose is converted to fructose
1-phosphate by fructokinase rather than fructose 6-phosphate by
hexokinase is explained in terms of the Km values of fructose and
glucose for the latter enzyme.
A possible general rationale for the two distinct pathways of
metabolism is to allow them to operate and be regulated independently
of one another. Although plausible, I am unaware of further data
support this idea.
The manner in which the fructokinase pathway evolved is a matter for
conjecture. I discuss what types of approaches to this question may
be valid, and the types of arguments that I consider invalid.

“Why?” Questions — A Note of Caution
Before addressing this question I think it important to consider what is meant by a “Why?” question in biology. This can be a trap for the unwary if one is not clear whether a question is answerable objectively or only speculatively. There are two main types of “Why?”questions:

Mechanistic “Why?” questions. For this SE Question this would seem to be: “What is the mechanistic explanation of the fact that
fructose is converted to fructose 1-phosphate, rather than to
fructose 6-phosphate, in liver?”
Rationalization “Why?” questions. In this case it would seem to be
“How can one rationalize the fact that a separate pathway for
fructose metabolism exists?”

Mechanistic questions can be answered objectively if there are sufficient experimental observations. Rationalization questions cannot.
Rationalization is helpful to try to make sense of observations — to provide a framework for further experimental work. It is useful in teaching and learning, which is why one encounters it frequently in text books. But just because it is in text books it should not be regard as on a par with the facts described therein. It is just an opinion, an interpretation, which should be regarded critically. There is a danger in accepting an inadequate explanation, just because it is the only one that has been proposed for something that is difficult to understand but we think that must have an explanation.
Rationalization “Why?” questions are often confused with questions about how something Evolved. These should be clearly distinguished. Rationalization explanations refer to the functional utility of a system, not its evolutionary history. If the only explanation is in terms of evolution, then there may be a plausable evolutionary explanation but no rationale.
An Answer to the Mechanistic Question
Fructose is not converted to fructose 6-phosphate in the liver because it has a much higher Km for hexokinase than glucose does. Furthermore, glucose is present normally at a higher concentration and will therefore act as a competitive inhibitor. Instead, as the poster writes, there is a specific fructokinase, which converts it to fructose 1-phosphate and a further enzyme converting to the fructose 1-phosphate to glyceraldehyde and dihydroxyacetone phosphate. The glyceraldehyde is phosphorylated to glyceraldehyde 3-phosphate.

Trying to Answer the Rationalization Question
“How can one rationalize the fact that a separate pathway for fructose metabolism exists?” The explanation given in the Encyclopedia of Human Nutrition — that it allows separate regulation of glucose and fructose utilization — seems possible, but fails to explain the circumstances in which it would be advantageous for fructose to be converted to pyruvate, but glucose to, say, glycogen. In the absence of this I would say that there is no convincing explanation, and that one should carefully consider a number of different possibilities:

It is chemically difficult to create a enzyme specific for fructose
to catalyse the formation of fructose 6-phosphate.  This seems
unlikely — we are not dealing with two molecules as similar as serine
and threonine.
Fructose 1-phosphate has functions (metabolic fates or regulatory
roles) other than energy generation in the latter stages of
glycolysis. The Kegg pathway map doesn’t suggest anything in the
former respect, but the entry in the Encyclopedia of Human Nutrition
states: “As fructose-1-phosphate is formed, it feeds forward and enhances the activation of pyruvate kinase thereby facilitating the passage of fructose carbon to pyruvate and lactate.” I do not have access to the book, so cannot say whether this argument is developed further. However as pyruvate kinase is also activated by fructose-1,6-bisphosphate (generated from fructose 6-phosphate) there would seem to be no regulatory difference between fructose and glucose metabolism in this respect.
Fructose 1-phosphate lacks some regulatory role of fructose
6-phosphate (or fructose-1,6-bisphosphate) and hence results in
different flux of fructose and glucose through glycolysis. This is
the converse of the previous possibility. It is true that
phosphofructokinase is activated by fructose 2,6-bisphosphate, which
is formed from fructose 6-phosphate, and so there is a feed-forward
effect when the concentration of fructose 6-phosphate is high.
However the fact that fructose 1-phosphate does not have this effect
would not seem to have any significance as it does not use
phosphofructokinase. Another possibility is proposed in the
Encyclopedia of Human Nutrition: “Since fructose enters the glycolytic pathway [later than the phosphofructokinase reaction] it does not exert an inhibitory effect on the gluconeogenic rate-limiting enzyme, fructose-1,6-bisphosphatase” As the only
relevant molecule that regulates fructose-1,6-bisphosphatase is
fructose 2,6-bisphosphate — inhibiting it — the argument would seem
to be that the entry of fructose into glycolysis through fructose
1-phosphate allows more glucose production for other tissues. Why
this would be a “good thing”, however, is not clear to me.
Unphosphorylated glyceraldehyde has other roles in metabolism, so its
production independently of glyceraldehyde 3-phosphate is more
energy-efficient. Unfortunately the other metabolic fates of
glyceraldehyde are specialized and minor, and do not seem to support
this possibility.

In conclusion, I personally cannot see a rationale for the separate pathway of fructose metabolism to pyruvate, although I believe that there must be one.
The Evolutionary Question
Let us use the question to consider arguments in this area. The approaches that I suggest may — in principle — be valid are those relating to actual mechanism. One can consider the number of evolutionary events that need to occur for a new pathway to evolve — the more steps, the more difficult. (I have used a similar argument in another SE answer). In this question the implicit argument is:

“The evolution of the pathway from fructose to glyceraldehyde
phosphate via fructose 1-phosphate requires three steps, whereas that
for a highly specific enzyme that converted fructose to fructose
6-phosphate would require only one.”

If one accepts this argument, it would suggest that there is some rationale for the fructose 1-phosphate pathway that has escaped us. Although this also seems likely for other reasons, one needs to ask whether the argument is correct. Did three separate enzymes really evolve to accomplish this, or were perhaps some of the enzymes already present and serving other purposes?
Two examples of common type of argument that I think are invalid are in the original question.

“As there are several disorders that arise from the deficiency of
enzymes used in fructolysis, wouldn’t there be a selection pressure
against two separate metabolic pathways?”

Selection pressure against causes of a human disease require them to have a severe effect on the survival of the species, to cause death before the individual is of a reproductive age, and to be relevant to early humans. Minor enzyme deficiency disorders of fructose metabolism, or even disorders arising from modern over-consumption of sucrose would exert absolutely no selective pressure. As it turns out fructokinase (EC 2.7.1.3) is widely distributed in nature — If one looks up fructose metabolism in fruit flies on KEGG one can follow a trail to a long list of species in which the enzyme is present. The point that I think this demonstrates is that for biochemical evolution, one needs to think when and where the pathway might have evolved.
The second example of argument that I think are invalid is

“Wouldn’t it be more economical to use a single reaction, so
everything would be easier to regulate?”

I refer to this type of argument as “playing God” or “second guessing Nature” as to me it presumes that one is in a position of such knowledge and wisdom that one is able to propose the optimal design for life.
Economical in what sense? Is our concept of economy the over-riding feature driving mammalian evolution? Multiple reactions and isoenzymes abound in the metabolism of animals. Apparently ‘wasteful’ energy- or material-consuming reactions like splicing are everywhere. And the regulation of intermediate metabolism is mind-boggling in its complexity. Regulation clearly evolved after the pathways, and “ease” of doing this does not seem to have been a driving force in evolution.
The lesson I draw from this is that even the most knowledgeable molecular scientist is in a poor position to say what is economical or efficient, and to predict the outcome from the cost/benefit tradeoff Nature will make or has made to do things “better”. And it is difficult to say what is “better”.

all_licenses: Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/


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Phosphorylation-Dephosphorylation : ATP-driven VS spontaneous
Is protein dephosphorylation most commonly spontaneous (without need of ATP)? I came across some papers that mention it, as well as the opposite case (ATP-driven), but I can't figure out which is common or which is rare.
In the case of protein phosphorylation, I would say ATP is always needed since it provides the phosphate? Or could you have a spontaneous phosphorylation using free phosphates that float around?
I am aware that in the end, pretty much everything is possible, so I am more looking for likelihood. What is commonly accepted, wide spread, and what may happen but is rare.

Well of course why not. Look at ATP, which is not a stable molecule, because it can spontaneously dephosphorylate.  Whether a phosphate group can be spontaneously hydrolysed depends on the nature of the molecule it is a part of. Are you asking about protein phosphorylations? In almost all cases that I know of, dephosphorylation in not ATP dependent.

Yes I am asking about protein phosphorylation.

Do you know of cases where dephosphorylation is ATP dependent. I certainly don't. You can add that info in your question.

I'd rather say google returns some articles, like this one. Again, It is hard for me to figure out whether this is well established science or not.
http://www.ncbi.nlm.nih.gov/pubmed/21401736

This is fine. Can you add this example in your question?

First, I would like to clarify what is meant by "spontaneous". In chemistry, thermodynamically favorable reactions ($\Delta G < 0$) are indeed termed spontaneous because they will eventually occur, regardless of how slowly. But if transition states are unfavorable (high energy barrier), the reaction rate can be extremely slow --- millions of years in some cases. For example, combustion of wood is highly favorable and, chemically speaking, it is spontaneous. But that doesn't mean trees are unstable and likely to catch fire any minute.
Most biologists would probably call a reaction "spontaneous" only if it actually happens on a reasonable time scale, perhaps on the order of hours, depending on the context. So the terminology is a bit messy. But neither chemists nor biologists would call a molecule "unstable" if it has a reasonable half-life, I think.
Now for the actual questions. Dephosphorylations are typically not ATP-driven, because breaking the P-O bonds is usually favorable already (although I'm sure there exceptions). However, the reaction is typically too slow to be useful to cells, so it is not "spontaneous" in this sense, and therefore needs to be catalyzed by phosphatases. A typical example is alkaline phosphatase which dephosphorylates a wide range of proteins and other substrates.
Phosphorylation requires a phosphate source of course, but also an energy source, because forming P-O bonds is not a favorable reaction on its own (the reverse of the above). So adding a lone phosphate would not be favorable, but transfering one from ATP is much better, because this simultaneously involves breaking a P-O bond in ATP, "freeing up" energy to drive the reaction.
Finally, an interesting point is that ATP itself is actually a quite stable molecule, with a half-life of about two weeks. So ATP hydrolysis is not "spontaneous" in the biologist's sense, and enzyme catalysis is required (kinases). In fact, the combination of a high "energy content" and good stability is probably key to the role of ATP as an energy currency in cells: if ATP was indeed unstable, it would randomly phosphorylate everything. Stability means enzyme catalysis can be used to control phosphorylation events. Truly unstable compounds are never used by cells, because their reactivity causes damage and toxicity.


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Typical in-vivo protein concentration
I am studying RNA-binding proteins involved in RNA granules. I am searching the literature for an order of magnitude of the absolute global concentration in the cytoplasm. Although I am aware this quantity can vary greatly from proteins to proteins, I would be happy if I find some papers having estimated it for some given proteins.

I guess you would want to exclude cytoskeletal proteins

Hard to find data for eukaryotes. For E coli some of these things have been measured and rough numbers have been made. I recommend the BioNumbers database as a great starting place: http://bionumbers.hms.harvard.edu/default.aspx . In particular, look up the (famous) table linked to by Bio Number ID 102990. It lists that at exponential phase E coli has about $2.35\times10^6$ protein molecules of 1850 types, so about 1270 copies of each protein. E coli is about 1 $\mu$m$^3$, so that's a concentration of about 20$\mu$M per protein if my math is right. But that's bacteria which are very different.


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Could a sperm be altered to contain a female's genetics?
While discussing with a friend a while back on the likelihood a futanari (a woman with both fully developed and functioning sets of genitalia) existing in real life, we got into a discussion of whether a child could carry the genetics over from 2 months.
Currently (to my knowledge), for a female same-sex couple to conceive a child and for both women to be involved, one would donate an egg, the other actually gives birth, and the sperm is donated by a third party. However, this means that the child will carry over the genetics of one women in the couple and the unknown male.
I am wondering if there is a way for sperm to contain the genetic information so that way both women in a same-sex relationship could be genetically related to the child.

Only as a comment for the moment: I remember to have read about animal experiments were it was tried to get embryos from male-male and female-female combinations. These embryos died very early due to the different imprinting of the genes and their possibility to get activated. Obviously some genes are activated from the mother while others are only activated from the father. If both copies are active or inactive due to epigenetic changes, this makes problems.

Note that some serious epigenetics disease might come up such as Angelman syndrome, Prader-Willi syndrome and Beckwith-Wiedemann syndrome.

There are now some papers and actual proteins that imprint the genome (due to mostly X chromosome inactivation) are found. So reprogramming the gametes' imprinting should now be possible. So now two gay man can produce a child. That is going to be interesting. There is also that chimera man who have different imprintings...

You might be interested in this article. In this study they created haploid ES cells from artificially activating an unfertilized oocyte and growing the haploid cells. These cells, when introduced in the ICM, give rise to viable chimeras.
In another study, haploid androgenetic ES cells were created by injecting a sperm into an enucleated oocyte. These cells also are capable of creating offspring when fused with another oocyte. [See this post also]
Also, since these cells can be cultured, they can be genetically modified. However, they undergo spontaneous diploidization and lose reproductive potential after a few rounds of division. This is related to the alteration of epigenetic status of these cells on certain imprinted loci. Others have also indicated the importance of both paternal and maternal imprinting in the viability of embryos which might be a problem in oocyte-oocyte derived hES cell fusions (See cross references). [Check this article too].
It can be concluded that oocyte$-$oocyte-hES fusions are not observed because of same imprint status (Also mentioned by Chris in the comments). AD-hES also lose their paternal imprints on prolonged culture (but retain maternal imprints). It is to be ascertained if paternal imprints are more unstable compared to maternal imprints under these conditions. It may be possible to optimize the culture conditions such that oocyte derived hES lose their maternal marks: would that lead to incorporation of paternal marks post-fusion is debatable.

Yes, it is scientifically possible. Though a third party (male sperm donor) is still required. Scientists working for a British biotechnology company, as well as doctors at Oregon Health and Sciences University have been working on complex procedures but are fighting the ethical red tape and are not currently using the techniques on patients.
This is the basics of the procedure being used by the doctors in Britain "The nuclei from the father's sperm and the mother's egg, which contain the parents' DNA, were removed. The nuclei were put into another egg from which the nucleus had been removed, but which retained its mitochondria. This new embryo contained the genes from both parents plus a tiny amount of mitochondrial DNA from the donor egg."
The scientists in Oregon have a similar yet slightly different approach with procedure explained as "Doctors would remove the nucleus DNA from the donor eggs and replace it with nucleus DNA from the patient's eggs. So, they would end up with eggs that have the prospective mother's nucleus DNA, but the donor's healthy mitochondrial DNA."
This approach can easily be modified to fit a lesbian couples desire to have a child that is related to both of them. Scientifically it is possible for two mothers to be directly related to a child, but the sperm is still a relevant part of the equation. It is not possible, so far, to create a functioning embryo without male sperm.
Sources:
http://digitaljournal.com/article/290608
http://www.huffingtonpost.com/2012/10/24/3-person-ivf-embryos-women-man_n_2011546.html
And my reproductive biology book and studies: Trends in Endocrinology and Metabolism by Claire Bourgain.

I don't think this is what was desired. This is used to mitigate the presence of mtDNA mutations in the female line. While technically the mtDNA donating mother has some genetic relationship to the child, this is so minor - only 13 proteins are coded for in the mtDNA,and they are all involved in the ETC. I think the question is looking for nuclear DNA from two females as discussed by @WYSIWYG

authors: Валерий Заподовников


text:
Why are my bacterial smears disappearing?
I'm trying to inspect simple stained bacterial smears. But my smear suddenly disappears after a successful inspection with the oil immersion lenses.
The background can become too red (the color of the stain) and the oil residue coming from the oil immersion objective when it's wiped, is red colored.

Red background
Usual background

Also when I try to wipe the oil from the slide, the smear is removed, even with a gentle wipe.
I'm suspecting three possible causes, which may include dependent causes:

The immersion oil I use makes the smear easy to wipe:
I don't have a dropper that releases just a small drop, but I have used another oil with the same type of dropper that releases more than the needed quantity and it didn't wipe my smear.

The back-and-forth movement that eliminates air bubbles: I hear the spring sound, is this correct?

Smear fixation may not be adequate: could it be possible that the smear can be fixed in an incomplete way so that it resists being wiped from the staining procedure, but not from further manipulation?

My specifications

Immersion oil: Non-drying, non-hardening Cedarwood oil

Stain: Carbol fuchsin (20%)

Smear fixation: heat fixation by passing the slide three times through the flame

Anyone else concerned about the fact it says "Ceder wood oil" instead of "Cedar wood oil"? (There doesn't appear to be a "Ceder Grove, N.J.") Also, "Conforms to ISO-8036/1 Specofication (sic)"?   -- I might recommend trying an immersion oil from a reputable supplier, one whose quality control for materials is reflected in the labeling.

@R.M. A quick image search on the internet appears to indicate that the labeling shown here is trying to imitate the general appearance of labels on genuine products of Cargille Laboratories in Cedar Grove, NJ.

@R.M. - That in itself is an answer. Their slides are mucking up because this is a knockoff product that's probably contaminated or simply contains something like cheap vegetable oil.

Bacteriological smears are a one-and-done scenario. Very few of them are intended for repeat use. Basically, all you can expect is that you observe it and then discard the slide. Wiping the oil off will also remove the bacteria as they are not firmly fixed to the slide, despite the "heat fixation" name, as I will explain below.
If you do want to reuse a slide I would recommend that you get a coverslip and cover your slides, sealing the edges with nail-polish or glue will make these more or less permanent. However, this is tricky because you may need to change your high power objective lens (assuming 100x objective) to cope with the thickness of the coverslip. To do this you will need to look for a longer focal-length on the objective lens. Some higher-quality lenses come with an adjuster for focal lengths.
Heat fixation in bacteriological slides is about two things. Primarily, the heating allows drying out of the specimen, which adheres it to the slide enough that it can be further manipulated. For most stains, simply drying the slide without heating will work perfectly well for adherence. Secondarily, it is about killing the bacteria. This is the fixation part and the name is a hang-over from classical biology where chemicals such as formaldehyde or ethanol were used to "fix" the specimen in an unchanging state (fixed state as opposed to changing).
It should be noted that even with passing the slide through the flame 3x, the slide rarely reaches above about 50 C, which isn't really enough to kill most bacterial species with certainty (food safety tells us at least 65 C for 10 min...), and is certainly not enough for tough bacteria like Mycobacterium species - it's the dehydration from the heating that does most of the killing. In addition, most stains have a fixative of some sort in them, usually ethanol, which will work against most of the common bacterial species (though also not Mycobacterium), again by dehydration of the bacterium, but also protein denaturation.

With the colour coming off, I think you have two problems:
First - staining, make sure that you wash the slide with decolourizer until there is no residual red from the Fuchsin. It's a delicate balance, so takes some experience to get it right. Also, make sure your slide is completely dry after your counterstain and wash step.
Second - I think that cedarwood oil is the wrong choice for immersion oil here. The cedrol and cedrene components strike me as chemically similar to phenol, which is one of the major components of Carbol Fuchsin (Ziehl-Neelson) stain (PDF with components). This means it might well solubilize the stain.
Cedar oil also hardens on the lenses and can easily dissolve the glues that hold the lens together, so be very careful using it with modern lenses.

I'm not doing an acid-fast stain but rather a simple stain if that would make a difference.

@FreezingSoul I don't know off the top of my head, but I suspect you won't get proper clearance of the slide without the acid wash, so will always have some residual red that might come off in the immersion oil.


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How does LCR compare to Assembly PCR
The question pretty much explains itself. How do the two methods compare? I've always used Assembly PCR but the method is prone to mistakes and I'm curious how it compares to Ligase Chain Reaction (LCR).

I am looking at this myself for gene synthesis.  OpenWetWare has a link to discussing the two.

In any PCR reaction ( including  the assembly PCR ) the substrate of the reaction is a mix of mono nucleotides. Specific oligos serve as primer by annealing on the template and that are extended by a DNA polymerase. It is a polymerization reaction.
In the LCR there are no mono nucleotides instead you have many different oligos that anneal one after the other on the template and a DNA ligase is used to join them together to form a longer DNA molecule. It is a ligation reaction.
This two reactions have much in common. Firstly, they rely on the annealing of oligos on a template. Secondly, both the polymerase and the ligase need to be thermostable. In fact, both reactions leverage the thermostability of the enzymes to carry on cycles of annealing, reaction (polymerization or ligation) and melting. After each cycle the template DNA is copied so you end up with an exponential amplification of your template with both techniques.
Here a nice presentation that showing these concepts with some graphics.

I have used both Gibson assembly (what you mean by assembly?) and LCR as replacements for blunt or sticky ended cloning. LCR has, for me, worked better in that a higher proportion of constructs that come out have been assembled correctly and I have noticed no mutations after LCR while I have seen them somewhat often with Gibson.

Welcome at Biology SE! Your answer is greatly appreciated, but on this site we prefer answers backed up with scientific references, Please provide objective references (if possible) to support your answer.

Thanks for the clarification Nandor. Also it would be much more helpful if you provide quantitative information when you are quoting your own data rather than 'higher proportion' - this would help viewers make their own judgements about which method they would want to use.


text:
How do you store membrane proteins?
We're producing some membrane proteins and they aren't amenable to freeze thaws even when we add glycerol. The proteins are solubilized in detergent above the cmc so they should be in micelle form in solution. Currently, they are being held at 4C. I'm curious what is the best way to store them for long term use?

this sounds like a case by case issue,but i'd be interested to see what someone with experience would say.

Can't you just store the cells you use to produce the protein and thaw them when you need them to produce more? Like that you wouldn't need to store the protein itself for a longer time.

You could always just add sodium azide and keep them at +4, so they don't have to go through a freeze/thaw at all.

@Suvidu, I would counter that if the purification protocol is a pain, it's a valuable investment to be able to scaleup effectively.

I've heard some MPs like lac permease can be scaled up, others are more delicate.  do you have an assay, or even crude crystallization conditions? that would really help of course.

I work with membrane proteins and in my experience some proteins can be frozen and thawed without any problems, other proteins just don't like that. It's linked to the stability of the protein. If you really need to freeze the protein you may think about playing around with different detergents to see if you can improve the stability of the protein.
General rules:
- freezing membrane proteins at -80°C without glycerol
- avoiding protein freeze-thaw cycles, making aliquots before freezing
- keeping them at 4°C only for short term use
- after concentration always spinning the protein (10 min at 100k for example) to precipitate aggregated material. Aggregation causes aggregation, so you want to get rid of it.

Hello metionina. This is an informative answer. Can you, if possible, add reference to the protocol that you follow.

I couldn't find any published protocol, probably because every membrane protein behaves very differently. This is what I learnt working with membrane proteins and what people mostly do where I work. Then, again, every membrane protein is different and I suppose you could find what I said true, partially true or false for your protein. These are for me general things I would do when working with a new protein.

If i were to have a blind attempt at your question, it is never a good idea to freeze thaw any protein, not even cell lysates. Thats the whole point of glycerol use, which we use in our lab at 50% (v/v) to store bead bound proteins. Thats also why antibodies and restriction enzymes are stored in glycerol since freeze thawing antibodies dissolved in water often leads to their degradation, although you only tend to store stuff in glycerol, like restriction enzymes, if the concentration of the protein is very very high. One golden trick I use is aliquoting my samples and snap freeze them in liquid nitrogen and store in -80 freezer. I'm curious as to what happens with your protein, does it degrade after a freeze thaw cycle? (I'm aware that transmembrane proteins are notorious when it comes to storage/maintenance due to their awkward hydrophobic transmembrane region so its best to speak to an x-ray crystallography expert, if there is some one like that roaming around your research lab :), since they have face problems like this quite frequently and have excellent suggestions). Are your proteins GST tagged and bound to glutathione agarose beads? or you use HIS tags and nickel beads and elute the purified protein through imidazole? i suspect it is GST in which case its probably not a good idea to snap freeze although I have (somehow) had frozen glycerol beads at -20 and they were fine after use. I have had a friend talking about protein purification and the fact that protein isolation has to be done fresh so you might have to go down that root if your current method is not ideal. Also one last question, are you purifying you protein bacterially or they get secreted into the media? I suspect if its a membrane protein you are using the cell based system since if it was E. coli you were using (as suggested above) you could have just had your protein expressed through IPTG and just washed you cell pellet in STE buffer and spun it down hard and stored the pellet at -20, in which case would save you some time in the purification process. The use of Na azide is a good idea too but I only use it for primary antibody storage which is already dissolved in the block buffer such as 5%-BSA-TBST for WB and store the whole thing at 4 oC and Na azide is there to prevent BSA and the Ab going off.
The main problem many many people have, including my self is that the majority of times, over expressed protein is insoluble or just doesn't get expressed in certain systems so you are very lucky to have a working system.
This is all I know about protein storage. I hope this helps.


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Removing bacterial contaminants from resin
We have a few Strep-tactin columns that had some growth in them and we would like to regenerate the columns back since the resin is quite expensive. Basic goal: remove the brown stuff.
So far, I've killed whatever living material there is with 20% EtOH and 0.5 N NaOH. After cleaning out the columns with Tris Buffer, I've tried regenerating the columns with Buffer with HABA and the resin is a bit redder than before (typical reflection of good regeneration). However, rinsing out the columns again still leaves behind a brownish color. There is also a good deal of black particulate in the resin.
I'm curious what methods exists to clean out a contaminated column.

I'd check the data sheet of the column for the limits of what the column can handle and just try the harshest of those conditions. They usually also specify cleaning procedures there. I'd try something with high salt to wash out that stuff, maybe also reverse the flow direction and run the column backwards.

I'm going to try the high salt. That's a good suggestion. The reverse flow probably won't work since I've repacked the column a few times by now

in the future consider azide is also sometimes used to keep columns sterile in storage.  Though it won't clear out the dead matter...its possible that the column was never meant to be this cluttered up.  In my wet lab days i'd be throwing that out... sorry to be a party pooper, but molecular purity is hard enough without whole cell fragments drifting about.

@Shigeta, alas it wasn't my column. I guess it's time to buy some new resin.

Yeah sorry, but remember your time is valuable too - don't spend 3 days trying to fix equipment that might ruin your prep...unless you want to be fixing equipment of course.

I am an engineer by training. The default option is always to fix it yourself. Usually with duct tape.


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Can humans' ability to dive with only breathing modifications tell us anything about our evolution?
Can humans' ability to dive with only breathing modifications tell us anything about our evolution?
For example, it is possible to imagine a human anatomy that would be destroyed by the pressure of a few meters of water, and yet humans can, with scuba apparatus dive to several hundred meters.

Actually, there was a very interesting Animal planet episode (http://www.youtube.com/watch?v=Cm3XdC-p8_I) on the recent news about the probable discovery of a mermaid body. In the show, scientists suggest that may be our evolution has had some roots under water. First, our body structure is far more plastic and ergonomic than the structure of a monkey. We are great swimmers in our movements and as you mentioned - our ability to hold our breaths under water for a very long time - the record is about 20 minutes! We can easily control our breathing which is something usually connected with marine mammals. In difference of other animals, we are almost hairless. Just like other marine mammals, we are born with insulating fat distributed throughout our bodies. This keeps us warm in water. All this takes place in the "Aquatic ape hypothesis". This view claims that our alleged ancestors went through a watery stage in our evolutionary development.
Of course, although all those facts connect very conveniently, the theory has not been proven yet by experiments and is so far just an idea that should be considered and investigated more.
In addition, some people still have marks from gills as an atavism.

The animal planet "documentary" is fake: http://oceanservice.noaa.gov/facts/mermaids.html.

@kmm: this post on Skepticblog also covers the fake documentary http://www.skepticblog.org/2013/06/05/mermaids-pullllease/


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Why must a planet be in a star's "habitable zone" to have life?
I have seen many articles that talk about a star's "habitable zone" as the ring around it which is just the right temperature for planets to have liquid water, which is necessary for life. My question is, why does a planet need to have liquid water in order to sustain life? Who says that extraterrestrial life can't live off of some other kind of sustenance?

Nice question, I guess this could get really philosophical and head towards asking "what is life?" - it's just what we define it as yet you are right, extraterrestrial objects might exist that under other definitions could be called life!

@GriffinEvo Really? The current definition of "life" involves surviving using water?

@GriffinEvo What are you asking? I don't understand the word "life" to require the use of water to survive.

This is really just a side point because I started thinking about it a bit... I think finding life that survives well outside of the conditions we define as being necessary for life would force us to reconsider what defines life. Part of our definition of life (google "define life") is the ability to respire (a process within "metabolism"), this requires oxygen, but what if we find something that doesn't respire in a way we recognize? i.e. not using oxygen. Can we call that a living thing? (I think we would have to call it life)

Your question is not complete. I would suggest to slightly change the title to something like:
"Why must a planet be in a star's “habitable zone” to have life as we know it from our experience?"
You are right, when you are assuming that there could be another than carbon based life as we know it from our planet Earth.
There are many hypotheses that life could be based on Si or other elements.
But carbon is still the easiest element to create bounds with other
elements and carbon too. Why? Here is the answer from wikipedia Carbon-based life:

There are not many
  other elements which even appear to be promising candidates for
  supporting life - for example, processes such as metabolism - but the
  most frequently suggested alternative is silicon.3 This is in the
  same group in the Periodic Table of elements and therefore also has
  four valence bonds. It also bonds to itself, but generally in the form
  of crystal lattices rather than long chains. Silicon compounds are
  generally stable but do not support the ability readily to re-combine
  in different permutations in a manner that would plausibly support
  lifelike processes.

There is a great documentary from a physicist Brian Cox published by BBC this year called Wonders Of Life which deals with the basic questions how carbon based creatures were "born" on this planet ;) + other interesting things about life.
Brian talks there why the first organisms were created probably in water and how it could be.
Why water?
http://www.pbs.org/wgbh/nova/evolution/liquid-of-life.html

And why is water the best liquid to do the job? For one thing, it
  dissolves just about anything. "Water is probably the best solvent in
  the universe," says Jeffrey Bada, a planetary scientist at the Scripps
  Institution of Oceanography in La Jolla, Calif. "Everything is soluble
  in water to some degree." Even gold is somewhat soluble in seawater.
  (Before you get any ideas about extracting gold from the oceans, I
  should add that, according to Bada, the value of dissolved gold in a
  metric ton of seawater comes to about $0.0000004).

If you take a route that there could be a different form of life outside of habitable zone, then many things are possible. E.g. life on asteroids, ouside of solar systems, life in other dimensions, life based on silicon or some other elements, energy based life? Or even dark matter and dark energy based life or gods/God-like creatures and all kinds of sci-fi ideas ;).

Why does the fact that water is a good solvent make it necessary for life?

@Daniel It is necessary for the life as we know it on our planet. If you want to speculate then many other things are possible. But the scientists take in count the fact how the elements are spread through the Cosmos. And they came to the conclusion that hydrogen and oxygen are pretty common. E.g. you have ice on Mercury, Earth, Mars and on Jupiter's and Saturn's moons, asteroids, comets and probably many other objects ad planets in space. More about the importance of water is e.g. in the 1st or 2nd episode od Wonder of Life, when Brain talks about the "cell = batteries" and "waterfalls";)

@Daniel here is a summary of what the water is capable of and why it is so important for life (as we know it) and why astrophysicists are looking mostly for planets with water as a possible life candidate http://www.youtube.com/watch?v=7sA_JJ9gj2E Btw. the thing with God is exagerating, but everything else is a nice summary of some articles. So, if you want to watch a 10 min talk about how the water is good for life, watch that video.


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Which kind of drugs get absorbed through epidermis?
Some drugs such as nicotine can be administered through skin. I thought the layers of skin are designed to prevent in-flow of any chemical/germs. Not all drugs get absorbed in this fashion. So do drugs like nicotine have any specialities(such as a special chemical group) that fool the sentries of skin? A link to an article/site would be very helpful, or even a book recommendation.

Looks like size is the largest factor: https://en.wikipedia.org/wiki/Transdermal_patch

"...that fool the sentries of skin..." The skin doesn't have sentries. It doesn't know anything, much less possess the ability to recognize drugs and let them in or keep them out. Finally, bacteria are much, much, much larger than the molecules that comprise the drugs.

According to a review (Prausnitz2004), marketed drugs with a dermal route of administration tend to fulfil the following criteria :

Small molecular size (< 500 Da)
High lipophilicity
Small required dose (up to ~milligram)

This is a consequence of the structure of the skin (with a lipophilic stratum corneum, and low diffusion speed) and practical concerns (everything else equal, a higher dose would need more surface area).
Beyond "pure" diffusion, there has been much work on improving drug delivery through other means : chemical enhancers (ethanol or DMSO being classical member of this class), medical devices (microneedle patch, ultrasonic device), and other more exotic methods mentionned in the paper.
These criteria are also mentionned in (GoodmanGillman2006, chapter 26), with emphasis on concentration gradient being the driving force behind diffusion across the skin. This chapter also contain a list of commonly used transdermal/dermatological drug, and their use.


text:
Does blood carry metallic nanoparticles?
I find this particular chart about constituents of blood very informative and interesting. Looking through the different components, I see metals such as copper, zinc, etc. Are these only carried as ions or conjugated to proteins, or does blood carry nanoparticles of these elements as well?

It is very unlikely to have metallic nanoparticles in blood under normal circumstances.
Reasoning:

There are specific proteins that strongly bind metal ions: transferrin and ferritin for iron,  zinc transporters (Zrt) for zinc, metallothioneins as a catch all for most heavy metal ions etc.  These tend to be intracellular so most of the metal would be bound inside cells.  Any particles would be endocytosed and oxidized to the ionic form (probably in peroxisomes), then the metal would be bound to proteins.
Metal nanoparticles as well as free ions (of metals with more than one oxidation state) have considerable catalytic activity which is non-specific (and therefore quite toxic).  They can serve as a redox catalyst as well as generate ROS from oxygen.  That's probably why metal-binding proteins have evolved.
Lastly, iron in particular is a limiting nutrient for many pathogens.  Extracellular iron is very tightly controlled as part of non-specific pathogen defenses.  There is only exactly as little iron in blood plasma (bound to transferrin) as absolutely necessary to move it to the cells that take it up.  It's unlikely there would be any free iron, because there wouldn't be any fast, specific way to take it up the way the transferrin receptor does.

'll ask a follow up question: What happens when metallo-proteins get denatured(say by thermal degradation), do they drop their metal payloads as elemental metals? Or as ions that are then taken up by other constituents?

@user1155386 Eventually, I suppose they must drop they payload at some stage of being recycled, but simple denaturation may not do it for many metalloproteins.  At least one reference I checked (Campbell PG, Metal Ions in Life Sciences. 2009 Jan 19;5:239-77) says that heavy metals don't leak out of the denatured protein fraction of cells. Most of them would drop as ions if they do drop (the exceptions being metal in heme and in iron-sulfur clusters)


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Impact of increased sodium conductance at a neuromuscular junction
If trans-epithelial Na+ transport were to increase at the synapse(please consider both pre and post-synaptic membrane situations) in a neuromuscular junction, how would that manifest itself? My guess is that the increased sodium conductance would depolarize the membrane to a higher resting potential.

Would this translate to higher firing rate or result in more frequency of miniature end-plate potentials?
Would this also affect the rise time of action potential? Or its threshold?


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How it is possible that blood group shows that a person is not the son or daughter of their parents?
I see a family in which it has only one daughter. The blood group of the mother is AB positive and the father is B positive, but the blood group of the daughter is O negative.
When she was young, they apply to shift abroad permanently. When the Embassy test their blood group, the doctor said "She is not your daughter because of her blood group".
How is it possible? Why is she not the daughter of their parents?

Have you tried reading from wikipedia > ABO blood group system? If yes, was there anything specific that was unclear to you?

yes because it is not clear for me thats why i ask this question i am confuse that how it is possible that a girl is not a daughter of her parents .why?

Your comment make it unclear what is unclear to you. The girl is the daughter of her biological parents (by definition). However, the people she calls parents might not be her biological parents. Genetics (in this example, ABO groups) can help to figure whether the supposed parents might be the biological parents.

@Jj Mmmm: How is it possible?  Obviously, either she was adopted, or the mother got pregnant by someone other than the supposed father.  See e.g. Homer and "It's a wise child that knows its own father".

@jamesqf: The mother is AB, so based on our normal understanding of blood types (without exceptions mentioned in my answer), she cannot be the biological mother of a child with blood type O.

Each person has two alleles for the blood type. The two A and B alleles are dominant. So, you don't need A/A to end up with blood type A, it could also be A/O. Having both A and B results in blood type AB. Having none of these, results in blood type O.
Following these rules, from parents being AB and B (genetically either B/B or B/O) you would expect these possible blood types for their children:
- A/B
- A/O
- B/B
- B/O
The Rh factor (positive or negative) is inherited similarly. The positive allele is dominant, so genetically +/+ or +/- would be positive, only -/- is negative.
There is a rare case of a "cis AB" mutation, where the A and B antigens are in just one allele (wiki, paper). Then the genetic alleles of an AB type could be AB (in one allele) and O.
The children of the couple could then be
- AB/B
- AB/O
- B/O
- O/O
So, there would be two possibilities for the case you describe.

The parents are not the biological parents of the child.
The mother is cisAB/O +/- and the father B/O -/-

edit: or a third way would be of course some de novo mutation in the child, resulting in the loss of A/B from the mother
edit2: maybe to answer your actual question "How can a child not be the child of its parents?".. adoption, in-vitro fertilization with donated eggs and/or sperm from other people, mix-up in the hospital..


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What is this ornamental, yellow flower that looks like a smooth pineapple skin?
My mom got this in her Mother's Day arrangement and we have no idea what it is.

Welcome to Biology.SE. As it is ornemental, the question might be a better fit on Garderning.SE.

@GerardoFurtado See the last paragraph of Madscientist's answer. I didn't remember the question, and wrongly said valid 'discussion', I'll remove it now.

The issue is not the fact that the plant is ornamental, but the fact that identifying cut flowers makes it impossible to define the "localization" of the species. For anything grown in the garden, we would at least know that it can grow in that specific location. Here we have very little to go with (even if it could be identified successfully this time).


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What is this 2-3cm insect found in South-Eastern Brazil?

Noted in South-Eastern Brazil rainforest at low level - about 2-3 cm from head to tail.
Maybe a Cassadine Beetle?


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Why do diseases in the tap water of developing countries affect people from developed countries more?
My siblings and I went abroad to a country that doesn't have drinkable tap water, but we did not know this at the time as the people who lived there used to drink it all the time with no issues.
One day, we all decided to drink a glass of water from the tap and we all ended up in a hospital and were really ill (I'm unsure of what we had as it was a while ago). We were told it was due to the tap water and were advised to only drink bottled water in the future as the tap water is unsafe.
My question is, why did the tap water only affect us but was fine to drink for the residents there? Is it maybe because our bodies are used to clean tap water?

The locals develop immunity to whatever is in the water, so they can drink it with no (or relatively few) problems. Since you didn't have this pre-existing immunity, you got sick.

So does this mean that certain pathogens in the water could in fact kill me but not others who are immune to them? Also does this mean that they did in fact get ill but a long time ago and then became immune after that? Also does this mean that I could be immune as I had the water and healed but with the help of some fluids that were injected into me. If certain pathogens in water kill humans how could someone become immune to it if they die when they have it? Sorry for the thousands of questions I just find it hard to understand. Thank you for your comment :)

Just to add to the other answer, people in those countries do still get sick on occasion and you don’t build up immunity to all possible pathogens. Getting sick now and then is just fact of life. I spent 5 years in SE Asia and locals would get the runs just as well. They just tend to blame it on other things like ‘spicy food’.

@James: Many different things at work, here. You can become immune to pathogens. You can develop a tolerance (which isn't the same thing). Or you can be smart enough to filter and / or boil the water before drinking it...

@AndrewT. Singaporeans had a higher chance compared to whom?

You can expect that someone drinking from a water source regularly will develop immunity to the pathogens in that water source by being repeatedly exposed to them.
However, it's not quite right to say that poor quality drinking water impacts mostly tourists. It mostly impacts the people drinking that water, especially children. Immunity comes at a cost. Some example statistics from Wikipedia:

Between 2000 and 2003, 769,000 children under five years old in sub-Saharan Africa died each year from diarrheal diseases

In South Asia, 683,000 children under five years old died each year from diarrheal disease from 2000 to 2003. During the same period, in developed countries, 700 children under five years old died from diarrheal disease.

And from WHO:

Contaminated water can transmit diseases such diarrhoea, cholera, dysentery, typhoid, and polio. Contaminated drinking water is estimated to cause 485 000 diarrhoeal deaths each year.

These statistics conflict a bit, and I won't go too far into the reasons why or try to identify the correct number. I think they are all sufficient to identify this as a substantial issue in the world.
Responses to disease vary between individuals for all sorts of reasons, including genetics, overall health at the time (for example, someone well-nourished versus poorly nourished), and access to medical care and treatment. Some you could also add up as effectively "luck". In a hypothetical scenario, you could expose 100 people to a pathogen, and of those 100, many may get very sick and a few die. The survivors may then have future immunity to the same pathogen, but it would be wrong to say the population as a whole is not affected by it, you're just looking at the people who survived.

Assuming that contaminated drinking water is the cause of ~25% diarrheal deaths (the majority stemming from contaminated food perhaps), the statistics need not be in conflict.

Looked at another way: Later repeated exposures to some source of pathogens are less likely to affect you than early exposures, since you’re more likely to be immune. So later exposures will bring your lifetime average risk-per-exposure gradually down (and your “recent average” more so), but will continue to add to your lifetime total risk.  So tourists have significantly higher marginal risk per-exposure than local adults; moderately higher liftime risk-per-exposure; and much lower lifetime total risk.

"will develop immunity to the pathogens in that water source by being repeatedly exposed to them." Either that, or they stay ill almost constantly and/or eventually die. Death by unclean water is still a common problem in third-world countries.


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How do trees communicate in the "Wood Wide Web"?
I read on The New Yorker - The Secrets of the Wood Wide Web that trees communicate with each other through electrical impulses sent through their roots. Through these impulses they can ask other trees for some nutrient if they are getting it in insufficient amount or warn the other trees about an upcoming pest attack. This phenomenon is termed as the Wood Wide Web.
So how do trees distinguish in different types of impulses that they receive if they don't even have a brain? And does this process show any resemblance to nervous system in animals?
Additional YouTube video: Trees can talk to each other, but how?

Welcome to SE Biology. It is a good idea to do the guided tour before you make your first post as it will familiarise you with how the site works. (You also get a badge, which is how I know you haven't). As a non-botanist I would still be very sceptical about the proposition in your question, so I would wish to see the evidence for it. You should therefore include a link to where "online" you read about it. Otherwise it may be closed as "unclear".

@David I have included 2 links one of a YouTube video on the topic and another link which I saw. Thank you.

Good. As I've put a space between your main question on the link to improve presentation.

Nowhere in your clickbaity source article is the word electrical found.

@Mindwin https://www.icr.org/article/7700 here see this link

Why do you think a brain is required to interpret communications? Single-celled organisms communicate with each other using chemical signals. And organs within larger organisms communicate using hormones and nerve impulses, but the organs don't have brains of their own.

Since it is very much relevant to the context here, sharing a book which has a lot to offer about trees and how they communicate and socialize.
The Hidden Life of Trees: What They Feel, How They Communicate—Discoveries from a Secret World by Peter Wohlleben I read it sometime back - a lot of interesting insights and thought provoking - and makes you realize why trees and forests are important part of human life.

This does not provide an answer to the question. Once you have sufficient reputation you will be able to comment on any post; instead, provide answers that don't require clarification from the asker. - From Review


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Viral vector vaccines - why doesn't the viral vector get attacked by the innate immune system?
I've been looking at how the Astrazeneca Covid-19 vaccine works:
A chimpanzee adenovirus (the viral vector) is injected into the patient.
After entering a cell, the viral DNA is deposited in the host cell nucleus.

The viral DNA only contains information about the SARS-CoV-2 spike
protein.
The viral DNA does not integrate with the host cell DNA.

mRNA is transcribed from the viral DNA, and this mRNA is translated into the SARS-CoV-2 spike protein.
This is displayed on the surface of the cell where it is recognised as an intruder by the immune system which then begins producing antibodies.
Pre-existing immunity is recognised as a potential problem. From Medical News Today - COVID-19: How do viral vector vaccines work?:

With all viral vectors, one issue to consider is pre-existing immunity.
If a person encountered the virus that serves as the vector in the past, they may have antibodies to the virus. This means that their body will try to fight and destroy the viral vector, potentially making a vaccine less effective.

But where is the innate immune system in all this? My (layman's) understanding is that the very fact that the virus particle was novel would result in an immediate response from the innate immune system. How does the viral vector get past it?

These Chimpanzee adenoviral vectors do not bypass the innate immune system at all. In fact they are used precisely because of their activation of the innate immune system, which then results in greater immunity because activation of the innate immune system results in antigen presentation and then activation of the adaptive immune system.
The reason they activate the innate immune system is because these viruses do not have a seroprevalence, so they can only activate the innate immune response with an end result of antigen presentation. In contrast, human adenovirus vectors generally have a high seroprevalence (because we've all had colds caused by adenoviruses), so they activate the adaptive immune response against the viral particle (vector), clearing it before presentation of the novel antigen (in this case the CoV spike protein) can occur.
There's a nice commentary on the different mechanisms by which the ChAdOx1 (Astrazenica/Oxford) and the mRNA (Moderna/Pfizer) vaccines work at Nature Reviews Immunology. To quote the relevant paragraph (emphasis mine):

The AdV vaccines also contain inherent adjuvant properties, although these reside with the virus particle that encases the DNA encoding the immunogen. Following injection, AdV particles target innate immune cells like DCs and macrophages and stimulate innate immune responses by engaging multiple pattern-recognition receptors including those that bind dsDNA — in particular TLR9 — to induce type I interferon secretion8. Unlike AdV vectors, mRNA vaccines do not engage TLR9, but both vaccine formulations converge on the production of type I interferon (Fig. 1). Type I interferon-producing DCs and other cells that have taken up the vaccine-derived nucleic acids encoding the S protein can deliver both an antigenic and inflammatory signal to T cells in LNs draining the injection site. This activates S protein-specific T cells and mobilizes adaptive immunity against SARS-CoV-2

One of the problems with using a simian adenovirus vector like the ChAdOx1 vector is that now a large portion of the population will have some antibody response to the vector (as well as the introduced spike), so future uses of this same vector might not result in as high an immune response as we have seen this time around, when the population is almost entirely naive.

I believe that if you read carefully, you'll see that your answer and mine are consistent with one another.

@jakebeal - sorry, I misread your post- I interpreted it to say that the innate system is suppressed and by implication the adaptive is used because of your second sentence. I've edited my post to suit.

@bob1 Re "One of the problems with using a simian adenovirus vector like the ChAdOx1 vector is that now a large portion of the population will have some antibody response to the vector (as well as the introduced spike)". What stimulates the antibody response to the vector? Is it "the adenovirus's foreign proteins" as mentioned by Jake Beale?

@Naj exactly. Just the same as if you had an infection with the adenovirus.

@bob1 OK. Finally, do these "foreign proteins" present themselves on the surface of the infected host cell along with the spike protein? Thank you for your patience!

@Naj I think you need to review how the system works, with the correct information, it should tell you how the vaccine works and how the body responds to foreign proteins in the context of infection.

@bob1 - will do :)

The innate immune system has many different components to it. If we consider a natural virus, like the base adenovirus used as a vector used in the Oxford/AstraZeneca vaccine, then we can assume that the virus already has sufficiently effective strategies in place to deal with them.
In particular:

Physical and chemical barriers are irrelevant, given an injected vaccine.
Interferon and MHC pathways must already be either ineffective or slow enough to be secondary in the response against the adenovirus's foreign proteins, such that NK cells shouldn't be expected to change their efficacy.
The vaccine protein isn't expected to damage the cell membrane, so that pathway won't change either.
Inflammation and antibacterial pathways aren't triggered by presentation of an extra protein either.
Activation of the adaptive immune system through antigen presentation is exactly the sort of response that is desired.


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Is there a formal definition of `nature` in Biology?
I've been discussing with a friend about Earth Day and at a certain point came out the question

What do you call "nature"?

He said that he considers "nature" basically all matter, including plastics and all kind of man-made materials.
I said that as far as I understand, "nature" is the ensemble of all Earth's ecosystems, thus if something is not part of an ecosystem, it is not "nature".
Is there an official definition of what is considered to be "nature"?
If yes, what is it? If no, is there a reason?

Why was the question downvoted? Please leave a comment. I personally think it is a good question

@Remi.b I guess we'll never know, but I have seens on several SEs people that think a question to be "unanswerable" and downvote without thinking too much about it.

Is there a definition of "nature"?
I don't think there is any commonly accepted definition of "nature" in biology. To my experience, the term "nature" is actually relatively rarely used in conferences or peer-reviewed papers.
Why is there no formal definition of nature?
The concept of "nature" has been developed outside the field of science or philosophy. As often concepts in the popular culture are used without a proper, complete definition. Even if there is an vague intuition that correspond to a concept, it does not mean that there is any way to make an objective definition of this concept. I think the absence of formal definition of nature mainly come from the fact that nature as used in the population culture does not mean much.
Is the term "nature" used in Biology and with which definition?
I just did a quick review of the use of the term "nature" in peer-reviewed articles. It seems that the term "nature" is rarely used or only in the abstract r in the first two paragraphs of the introduction to convey very general (and somewhat inaccurate) ideas.
Often, the term "nature" holds for "essence", "origin" or "intrinsic characteristic" rather than referring to "outside the lab", "landscape" or "ecosystems". Here are some examples of where I found the term nature
"Nature" as "Essence"
From the abstract of Gibson and Dworkin 2004

[..] we highlight recent progress in determining the nature and identity of genes that underlie cryptic genetic effects [..]

From the first paragraph of Woolhouse et al. 2002

Failure to recognize the dynamic nature of the interaction could result in misinterpretation [..]

"Nature" as "outside the lab"
First sentence of the second paragraph of Elena and Lenski 2003

Since Darwin’s day, many examples of evolution in action have been studied in nature [..]

Note btw that there is no good (objective) definition of life either. One might want to have a look at Why isn't a virus alive? for a discussion on the definition of life.

The meaning of words depends on how people use them. However it would appear that your friend is in a minority applying the word ‘Nature’ to man-made things. Thus,
The OED definition includes:

The phenomena of the physical world collectively, including plants, animals, the landscape, and other features and products of the earth, as opposed to humans or human creations

and Merriam-Webster:

the physical world and everything in it (such as plants, animals, mountains, oceans, stars, etc.) that is not made by people


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Cardiac Excitation Threshold - in C++ modelling
So I am trying to write a code in C++ about the cardiac excitation threshold.
I know that this excitation threshold is the shortest stimulus2 value at which it can conduct an action potential (known as the effective refractory period) at that stimulus1 value.
So my question is: In my code should I have: apply_stimulus1 for basic_cycle_length = 1000 and while you are applying this stimulus1 apply and a stimulus2 and print out the shortest value of s2 where I can have a potential? -> the way I am thinking this is apllying both stimulus simultaneously. If we say .... this is stimulus 1 and this is ,,,, stimulus2 at the end I will have ;;;;.
Or should I say when you finish applying that stimulus1 apply a stimulu2 and give me the shortest time where I get action potential? But here I will have stimulu1 .... || (meaning ends applying stimulus1) ,,,, apply stimulus2.
Is this how I am thinking it correct or I just got lost in my own thoughts and it is easier than I make it look?
My question is:
How do I get the cardiac excitation threshold using stimulus1 and stimulus2 and how can I incorporate the theory in my C++ code.

ok..i just wanted the biology part first so I can give it a try in C++ and then I will post in stackoverflow, if that is ok..

Please post your question on http://stackoverflow.com/ for help in C++ programming. And you need to edit your question for more clarity. The way it is now, it is confusing.

This doesn't seem like a programming question, however, the basic point of the question is unclear.

i just want to know how two different stimulus can create cardiac excitation threshold. As Devon replied below it would be applying stimulus1 for a certain time then wait and then apply stimulus2. So as this question is answered how can I incorporate this in my code. Is it more clear now or still confusing?

I'm not sure how this is off-topic. This is a subject covered in an intro undergrad neuroscience course. Aside from the programming aspect, I'd use a topic like this for an undergrad exam question (e.g., "design an experiment to demonstrate the concept of relative and absolute refractory periods"). This is just taking that and actually writing a program to do it.

neither do I, but anyhow.. thanks @Devon Ryan for your help.

You wouldn't normally apply the stimuli simultaneously. The idea is to apply stimulus1, which has a given amplitude and duration, and follow that with stimulus2, which also has a given amplitude and duration, after a variable lag. You'll then see that the effective refractory period is dependent on the amplitude of stimulus2 (at least until you hit the absolute refractory period).

so I will proceed with the way#2 -sort of. For example apply stimulus1 for 1000msec then wait for 5msec and then apply stimulus2? But should I got backwards in time such that I can create APs at my last AP created by Stimulus1?

Yes, you'll apply stimulus1 for 1sec and then wait, say, 5msec and then do stimulus2. Keep in mind that 1 sec is probably far too long...you normally just want to stimulate a single AP afterall, not a train. I have no idea what you mean by "go backwards" in this context.

i was thinking like if my variable lag is 5msec would be easier to generate an AP from 5msec decreasing to 1msec until it gets to the last AP of stimulus1 (is it clear now? :S ) also it is in 1D strand so isn't better to be a train of  APs?

Ah, got it. You'll want to use a minimum lag (say 1ms or so, depending on you AP duration). Otherwise, you end up just looking at a larger stimulus, which isn't informative. By a train of APs, I don't mean an AP traveling down the compartments of your model, unless you have to elicit a train in the first compartment to get propagation. Anyway, you certainly can use a train, I just wouldn't bother without reason. I should note that the last time I used/wrote a model like this I did it with skeletal muscle, so if I'm forgetting that cardiac models should show bursts then go with the train :)


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Can a bacterium survive without GroEL protein?
In prokaryotes, GroEL protein (together with GroES) is required for protein folding.
Question: Can a bacterium survive without GroEL protein?

In E. coli, GroEL/GroES is found to interact with about 10% of all soluble proteins (Kerner et al. Cell 2005) and is the only chaperone essential to the bacterium under all tested conditions (Horwich et al. Cell 1993).

There doesn't appear to be a knockout for it in the Keio collection (they systematically knocked out genes in E coli to assess essentiality, via recombineering).
If they couldn't knock it out, that strongly suggests it's an essential gene and the organism cannot survive without it.
http://cgsc.biology.yale.edu/KeioList.php
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1681482/

There is a groEL knockout: http://cgsc2.biology.yale.edu/Strain.php?ID=109235

That's only a GroL knockout, so I'd say the point still stands.

I'm fairly sure groL and groEL are synonyms.

You may be right, I assumed they were a pair, but I think I'm confusing this with it's interaction with ES.
If so, I stand corrected.
And that nomenclature is stupid :p

Haha agreed. Also, in the page I linked to: "There is some question as to if this strain has a complete knockout of groL".

Yeah I know with the KC they've been plagued by false results.