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Replacing, instead of repairing, DNA
I've been doing some light reading on DNA damage theory of aging. One of the main ideas from that theory that I got is that the accumulation of damage in our DNA is one of the biggest causes of why we age, which is quite intuitive for a non-biology major like me. Furthermore, there has been research on DNA damage repair pathways and ways on how nanotech can help repair DNA. The technicalities are beyond me. My question is

If accurate DNA repair is such a difficult mechanism to sustain over a
  lifespan, then why not catabolize DNA and replace it with a new one?

It seems very feasible to synthesize human DNA (please inform my ignorance, if I'm wrong. lol.) and it looks very promising after reading this: https://www.wired.com/story/live-forever-synthetic-human-genome/.
EDIT: It was pointed out to me in the comments that my question is too broad, please do tell me how to make it more specific, if you agree that's the case here. I'm very happy to accept technical answers, since I can always retrace it to the basics.

By "we" do you mean nature or medical intervention?

"we" age due to nature...?

@TheLast_Cipher Excuse me, I meant when you said "why not catabolize DNA and replace it". My point is, are you asking why we've not evolved to replace our DNA or whether we could use DNA replacement as a medical therapy.

@Jam: I am asking why we have not use DNA replacement as a medical therapy.

It seems very feasible to synthesize human DNA (please inform my
  ignorance, if I'm wrong. lol.) and it looks very promising after
  reading this:
  https://www.wired.com/story/live-forever-synthetic-human-genome/.

It is certainly possible to synthesize human DNA, but it's another thing to create a functional chromosome. In fact, in the article you link to, they are discussing a research effort to synthesize a human Y chromosome. As far as I can tell they haven't actually done this yet, they are just proposing to attempt it. George Church is a highly regarded genomicist, but the article is very sloppy about distinguishing what they think they can do from what they've actually done. There has been work with human artificial chromosomes (HAC), but these have been special purpose constructs that successfully replicate and divide in cells, but don't duplicate an entire natural chromosome.
Synthesizing a human genome would just be the start of the therapeutic process. You then face the question of how to place the synthesized genome in the cells of the recipient. Current approaches to gene therapy involve inserting a single "repaired" gene into a virus, and then infecting the relevant tissue with that virus. This doesn't insert the gene into all cells of the body, just the ones infected with the virus. For some simple genetic diseases this is enough to relieve the symptoms of disease. You can't insert an entire human genome into a virus though.
It's also been proposed to harvest stem cells from the recipient, modify them with the repaired gene, and re-insert them in the diseased tissue. Again this would affect only a portion of a single tissue, not all the cells of the body. It's also one thing to insert a short sequence into a stem cell, but nobody knows if you could swap out a stem cell's entire genome and still maintain it as a stem cell. Also, this would only work for tissues that are still actively dividing. Many important tissues (in the brain for example) have stopped dividing or divide only slowly in adults.
Even gene therapy involving single genes has been approved in only a handful of diseases. It's sill almost entirely experimental. What you are suggesting is still in the realm of wild speculation. Speculation can be inspiring and motivates research programs, but don't starting counting chickens yet.

Is it correct to say that a DNA becomes a functional chromosome only when it is in an environment where it can be transcribed and replicated? So delivery methods are just as important?

@TheLastCipher it's even more complicated than that. Our chromosomes are complex structures with the DNA wrapped around a trellis-work of histones and other proteins. The 3-d structure of the chromosomes plays an important role in the regulation of gene expression and is controlled in turn by a hundreds of proteins coded in the DNA.

I would like to encourage further discussion so I'll retract the acceptance of your answer. Hope you don't mind.

@TheLastCipher you're welcome to retract the acceptance of the answer and vote it  up or down as you see fit. However do note that StackExchanges is intended to be a question and answer site, not a discussion forum, ala Reddit or many others. If you would like an open ended discussion on a topic you can open a chat room once you have enough points.


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What is the exact definition of mitochondrial depolarization?
As I understand as per literature reading and some khanacademy(https://www.youtube.com/watch?v=PtKAeihnbv0&t=315s):
Proton gradient := the gradient created when hydrogen ions moves to the intermembrane space from the mitochondrial matrix.
Mitochondrial membrane potential := the potential energy created by the proton gradient.
Does depolarization mean the loss of mitochondrial membrane potential? The lower the mitochondrial membrane potential, then the more depolarized the mitochondria?
So the way I defined proton gradient and mitochondrial potential is conceptually derived from the so-called proton-motive force used here https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2605959/ and also described here https://www.dummies.com/education/science/biology/the-proton-motive-force/ as an electrochemical force. Thus, my definition is from my rudimentary physico-chemical intuition.

What does “:=“ mean, and where did you get these definitions, which I would consider poor or incorrect? Where have you encountered the term “mitochondrial polarization? To be meaningful you should provide sources and context. If you have read one thing and providing your own understanding of other terms please make this clear.

Sorry, David. This is simply how understood it based from youtube videos and literature reading. So it is my personal interpretation. I posted it so people who have the expertise and who care to correct it, will.

Then perhaps you could modify your question to give a specific source for the use of the term so we can see whether it was used in relation to mitochondria as a whole or just the inner membrane. Also it would be better to rephrase the other sections as "I understand that the mitochondrial membrane potential is..."

Hello David, I've added some of the sources. Thanks!

Ok. I'll see if I can find anything of use to add.

The inner mitochondrial membrane is important for ATP synthesis (oxidative phosphorylation) to occur. Electron transport chains are situated on the inner mitochondrial membrane where electrons traverse the chain across proteins - causing protons to traverse into the mitochondrial matrix. Creating an electrochemical proton gradient.
This is a very interesting topic and is best understood with an explanation of the JC-1 Dye.
Explanation
Mitochondrial membrane potential changes - depending on cellular events.
The dye 'JC-1' is able to indicate mitochondrial membrane potential in different cells.

If there is a negative charge inside mitochondria then the monomers express green fluorescence.
If there is a positive charge inside mitochondria then the monomers form J-aggregates and now express red fluorescence.

The ratio between red & green florescence shows the mitochondrial membrane potential.

A low or negative mitochondrial membrane potential will show more green florescence.
A high or positive mitochondrial membrane potential will show more red florescence.

Example
One event that may influence mitochondrial membrane potential is an increase in reactive oxygen species.
Reactive oxygen species are molecules that damage cells but are also important to regulate cellular activities. Some examples are Hydrogen Peroxide or Superoxide.
In this image cells are stained with JC-1. The ratio of the dye is ~1:1 at basal. As the reactive oxygen species hydrogen peroxide is added and time continues, there is more green. Showing:

A low or negative mitochondrial membrane potential in each of the cells.

Thank you very much. This is helpful. This does agree with the assumption that ROS can induce "damage" to the membrane complexes; hence a low mitochondrial membrane potential, i.e. green fluorescent? May I also know the reference of the image? Lastly, what are your thought on the definition of depolarization?

@TheLast Cipher
"In general, harmful effects of reactive oxygen species on the cell are most often:[18]
-damage of DNA or RNA
-oxidations of polyunsaturated fatty acids in lipids (lipid peroxidation)
-oxidations of amino acids in proteins
-oxidative deactivation of specific enzymes by oxidation of co-factors" - ROS wiki page.
High levels of ROS changes the gradient.
Image Ref: https://www.thermofisher.com/au/en/home/life-science/cell-analysis/cell-viability-and-regulation/apoptosis/mitochondria-function/jc-1-dye-mitochondrial-membrane-potential.html

@TheLast Cipher
Apologies, I didn't have enough room.
A depolarized mitochondrial membrane potential is an indicator of increased levels of ROS. Ref: Thermofisher.com link above.

@Andrew — I have no interest in you or "everything you do", indeed I am generally not aware that I have commented on people's posts previously unless they have very distinctive names. My only interest is to improve answers (and questions) to make them more useful to the community. As you are new here, you may not realize that comments are primarily for asking for clarification or making suggestions for improving answers. That was the point of my remark. Let me reword it. "I suggest you could improve your answer by distinguishing between the potentials of the two mitochondrial membranes".

source: Stack Exchange

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

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Why do some people accumulate more diglycerides in their muscle cells?
The scientist Gerald Shulman has experimentally found that young lean adults in their early twenties that are children to people with type 2 diabetes often show muscle insulin resistance. He found that diglycerides within the muscle cells made it harder for the insulin activated "glucose transporter 4" to attach to the cell membrane.This causes these people to have raised insulin levels in order to get enough receptors to get glucose into the cells.
Also he has found that young adults of the same age and BMI that are not "sedentary" but more physically active do not have insulin resistance in the muscles.
Shulman believes that muscle insulin resistance due to diglycerides in muscle cells is what is starting a series of events that eventually can result in insulin resistance of the liver and diabetes. He has more then 450 published papers on related subjects and I can not really find "the one paper" where he claims to experimentally have verified his assertions.
This link is from a presentation from when he won the Banting Medal:
https://professional.diabetes.org/webcast/banting-medal-scientific-achievement%E2%80%94mechanisms-insulin-resistance%E2%80%94implications-obesity
There seems to be different theories as to why some people tend to accumulate more diglycerides in their muscle cells.
Question: What are the major reasons some people tend to accumulate more diglycerides in their muscle cells than others?

Remember the one question per post rule we have here? Asking so many in one post is liable to result in less than full coverage of the topic.

Further to @ARogueAnt., Asking so many questions is likely to have your question closed.  This is Q&A site, not a journal club or a discussion site. It might be worth your while looking at the Help on Asking Questions.

Thank you for focusing down to a single question, but I think you would get a better response if you supplemented the youtube links with references that supported the assertions you make. That would also demonstrate that you had done the expected research. ——— This is a good example of how to format references. Also note that tags should be created sparingly, should never be redundant, and are most useful when they apply to multiple questions.

Having already made a criticism of your question I am reluctant to make another, but please accept it as having constructive intent. Professional scientists have an aversion to individual scientists publicising their views on videos. That is not how science is done. Like @tyresome, I read peer-reviewed papers and look at the evidence, not the person. A search for "diglycerides and insulin resistance" brought up a number of papers on this topic, implying that the correlation is well known. I suggest you read these yourself to see if there is any work on the cause of increased DGs.

@david I can see that this is a very active field of research, some clinical trials of diabetes-oriented medicine regulating lipid metabolism seem to be coming up. Some researcher thinks that fructose consumption via some pathways destroys lipid metabolism and thus causes insulin resistance. I am trying to lift the knowledge in this area (which do not seem to be widely spread) to a wider audience. For that I need answers from people with higher ranking than me on this site.

Clinical trials don't give information about mechanism. Neither do researchers' thoughts. If diglyceride synthesis increases as a response to some change, the mechanism by which this occurs can only be elucidated at the cellular and subcellular level. There are not too many biochemist frequent this site, and this is a rather specialized area. Sometimes I would try to research the literature out of interest, but I'm not inclined to do so as I'm busy and suspect that it may be a wild goose chase. You might try on SE Medical Sciences if you get nothing here, despite your bounty.

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


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Antibodies combine with antigens in the presence or abscence of macrophages?
There was a mcq in book that antibodies combine with antigens when.... And the correct answer was if macrophages are absent. As far as I know macrophages release interleukin 1 which stimulates T helper cells to release interleukin 2 which further activates b cells. Plasma clone b cells then produce antibodies.  With respect to that macrophages presence is important.   Kindly clear my confusion.

Could you provide more complete context about what your book is saying?

It wasn't from the text.  It was just an mcq.

I suspect there is further context to the question that would help explain your confusion, without it I can't say much. If it's just completely isolated then I don't think it makes much sense as a MCQ by itself.


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What is tissue gel?
My book consists of following lines."The interstitial fluid is entrapped in minute space among proteoglycan filaments. This combination of proteoglycan filaments and the fluid entrapped within them has a characteristic of gel so called tissue gel.  Instead of flowing interstitial fluid mainly diffuses through gel.  This diffuion allows rapid transport of interstitium of water nutrients,oxygen,cell excreta e.t.c.
I can't get the meaning of these lines.  I tried to search on net but didn't get a satisfactory explanation.  Please explain this to me.

In the question, "tissue gel" clearly refers to "proteoglycan filaments and the fluid entrapped within them" and which appears in the interstitial space.
This is the same as what the Wikipedia article says about Extracellular matrix:

Gels of polysaccharides and fibrous proteins fill the interstitial space and act as a compression buffer against the stress placed on the
  ECM. (Source: Essential cell biology, 2004)

A.C, Guyton et al from University of Mississippi School of Medicine, 1973 also says the same:

Though 1/6 of most tissues is composed of interstitial fluid, this
  fluid is generally held tightly in place in the form of a gel. The
  reticulum of this gel is composed mainly of hyaluronic acid, but this
  is cross-linked slightly with collagen fibers to form a meshwork in
  which the interstitial fluids are trapped. The presence of this gel
  reduces the mobility of fluid in the tissues...

The exact term "tissue gel" is very rarely used in biology or histology, though.
You are probably confused because of this sentence, which is messed up: "This diffusion allows rapid transport of interstitium of water, nutrients, oxygen, cell excreta e.t.c. " It should be: "This diffusion allows rapid transport of water nutrients, oxygen, cell excreta e.t.c. through the interstitium."


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Is the "first pass effect" diminished more by some foods than others?
This effect and its considerations are fairly well known but it's never been clear to me whether it's a greater consideration with some types of food than others.

This completely depends on drug in question.

@Jan I don't see how. My question doesn't ask the extent to which it happens; merely whether or not it happens at all. If any drug that experiences a first pass effect has that effect diminished more by some foods than others then the answer to my question is "yes."


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Is there any checked case of "involution"?
I know that strictly speaking the term "involution" referring to the evolution theory doesn't hold any meaning. Organisms keep on evolving and changing with time, that's all.
But the following situation could perfectly happen:
Some event causes some organisms to evolve certain characteristics that allow for a better survival, but with time that event stops and what was previously and advantage for these organisms, now it's a disadvantange, and to adapt to the new situation what they do is to exactly remove these characteristics and they end up being exactly the same as they were previously to the change. It could be considered that they have "involutioned".
Are there checked examples of this? (Not just as theory, examples of organisms where this "involution" has been observed). I'm specially interested in "complex" organisms, I think this is not that far fetched to happen to organisms like bacteria, virus or the like. But for more "complex" organisms I guess it's too much of a coincidence.
Edit: I'm putting an example to clarify:
Let's imagine we have a huge amount of plants of the same species which can grow extremely fast (in the way they could simulate 100000 years in evolution in one hour) and we have an atmosphere for them that is the same as current one. (Not realistic, but let's make the thought experiment). We also put some put of these plants apart.
Little by little we go changing all the carbon dioxide in that atmosphere with something, like, for example, nitrogen, it's to expect, that, with time, some plants will appear to better use the nitrogen, at least up to the point where they can survive, as carbon dioxide is every time lower and lower and plants not adapted to this end up dying.
When we finally have only nitrogen processing plants we start the opposite, we start filling little by little with carbon dioxide, it's to expect that the contrary would happen.
Now, if a biologist were to observe the plants that were set apart at the beginning and the new created plants he wouldn't be able to tell where each plant is from, unless he was aware they were separated to do the experiment.

Are you asking about an example of varying selection pressure (with associated adaptation) over time? Something like "temperature decreases, creature evolve to be more hairy. Temperature increase back, creature evolve to be less hairy"?

@Remi.b, it's more than that, it would be creature evolves to be more hairy, creature goes back to be the same.

Isn't it exactly what I suggested? I had never heard of the concept of "involution". According to wikipedia it refers to the physiological change in organ size. It has nothing to do with evolution. You might want to just get rid of this term alltogether and just ask for an evidence of varying selection pressure (with associated adaptation) over time? Eventually, add a specific example (such as my hairy/hairless example) with it for clarification.

"Involution" is basically a made up word that doesn't hold any meaning in this context, although it's translation is used in my native language for describing behaviors that people make that are not supposed to happen nowadays, as if it were to mean that they are some previous organism. But well, we can forget about the word and I'll put an example that clearly explains it.

Yes there are several examples of retrograde evolution (traits present in ancestors but lost in a species). It can be subtle as loss of some non-central metabolic pathway (Vit-C synthesis) or drastically visible as loss of body parts (for e.g. limbs of snakes). I just saw an entire book on this topic. But there can be drastic trait losses in micro-organisms too (for e.g. loss of mitochondria). Is this what you are looking for?

Australian kangaroos were originally rather small marsupials which evolved into megafauna due to a changing environment. We are not certain as to why the megafauna went extinct, though we do know that their size was reduced to what it was previously, albeit some alterations in skeletal structure and such had occurred.

"Exactly the same" probably doesn't fly, just because there is so much possible variation, but for a fairly obvious example, consider birds.  Their ancestors evolved flight: some modern-day birds (ostriches, emus, kiwis, &c) have lost that ability, though their vestigal wings haven't re-evolved into legs.  But consider the evolutionary link between birds and theropod dinosaurs, and how many of those (e.g. T. rex) had vestigal forelimbs.
Another example would be dolphins and ichthyosaurs, which both independently "involved" from land-dwelling creatures back to a form that's similar to the ancestral fish.
But of course neither of these are really "involution" in the sense of reversing an evolutionary path.  They are cases of organisms exploring new evolutionary paths, where the environmental pressures (such as need for streamlining to move efficiently in water) cause them to resemble some ancestral form.


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What does "count in gene set" mean?
I am trying to understand the analysis generated by STRING.
In analysis for biological processes table, there is a column titled "count in gene set." This gives a value for example "68 of 498".

What does this refer to?

It means the software has 4753 genes it has labeled with "biological regulation" and the short list you gave has 45 of those genes.  That is a little higher than you might expect by chance if you'd given a random gene list of the same size.

2.94e-06 FDR suggests to me it is significantly higher than you might expect by chance.


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Given that "genetic drift" can happen, why is searching for an adaptive explanation for traits considered an important scientific question?
I saw today on Wikenigma (a wiki for open questions in science) that there are many competing explanations for why zebra stripes are evolutionarily adaptive, and there is no consensus on which one(s) are correct.
The discussion there suggests that scientists are eager to explain the specific adaptive advantage conferred by zebra stripes—for example, to find evidence showing that zebra stripes help the animals regulate their body temperature.
However, we know that there is a phenomenon called genetic drift by which certain genes come to dominate a population "just because": These genes are neutral with respect to evolutionary fitness, but happen to secure a majority early on in the lineage, and this majority grows over time:

Genetic drift is one of the basic mechanisms of evolution.
In each generation, some individuals may, just by chance, leave behind a few more descendants (and genes, of course!) than other individuals. The genes and other genetic elements of the next generation will be those of the “lucky” individuals, not necessarily the healthier or “better” individuals. That, in a nutshell, is genetic drift. It happens to ALL populations — there’s no avoiding the vagaries of chance.
…
Genetic drift affects the genetic makeup of the population, but unlike natural selection, through an entirely random process. So although genetic drift is a mechanism of evolution, it doesn’t work to produce adaptations.

Let's fix the meaning of adaptations here:

An adaptation is a feature that arose and was favored by natural selection for its current function. Adaptations help an organism survive and/or reproduce in its current environment.

(source)
My question is, given that "genetic drift" can happen, why is searching for an adaptive explanation for traits considered an important scientific question?
Non-questions

Zebra stripes are a motivating example for this post, but not the central theme. If you believe that zebra stripes do confer a specific adaptive advantage, then please substitute "zebra stripes" with a trait whose adaptive advantage you cannot identify.

I'm not sure that I see the justification for point 3, as that's the only place that you've specified that the father has some fitness advantage. Zebra stripes have a known adaptive advantage - did you look them up, what did you find? If something is preferentially selected for, then it is by definition a selective advantage, it doesn't make any difference if it's due to the feature being attractive or a good survival trait - see peacock tails as example of female selectivity of non-advantageous trait. Can you clarify?

Point 3 is mentioned in the preceding paragraph. Your first question is addressed by my first "non-question." Your second question feels like a semantic issue: you say "selective" advantage but I chose the word "adaptive" on purpose, because the search for an explanation for zebra stripes seems deliberately targeted toward adaptive explanations and nobody seems content to take "the stripes are attractive" as an answer—and again, the zebra stripes are just an example, and you can substitute peacock tails if you think zebra stripes have an adaptive advantage.

“Can natural selection reward traits…?” Please consider carefully the meaning of the words “selection”, “reward” and “traits”, and you will see that your title is scientifically and linguistically meaningless. Traits may be selected, and one might say that evolution involves the selection of certain traits. That is the only relation between the two. “Reward” is not a feature of evolution in any sense.

“ Now suppose that a strong, adaptively fit male of this species happens to be born with a heritable sexual preference for trait A.” 1. That favors reproduction, which means selection. You can’t make it neutral just by placing a label on it. 2. How do you think traits evolve? A male just happens to be born with an interest in pattern “s”? Why would that happen in one jump? Is preferring a metal vs. a wooden frame a trait that is heritable? Your words… don’t seem to particularly comprehensible, and they certainly don’t mean what you seem to believe they do.

for neutral traits look up something called "genetic drift".

I’m voting to close this question because it is unclear (this includes your explanation for why you think the linked page doesn't answer whatever you are trying to ask) and under researched. In particular, this appears to be based on an inadequate understanding of the basics of evolutionary theory — I encourage you to spend time reading and thinking about the material in this useful introduction to evolutionary theory from UC Berkeley.

I'm frustrated that many of the responses here seem to boil down to pointing out that I have used words in a different sense than evolutionary biologists do. I gave a clear example of what I mean by an "adaptively neutral" trait; while I understand that my example is technically "adaptively positive" in the sense that it favors reproduction, it is obviously qualitatively different from traits such as opposable thumbs that confer a specific biological advantage, and this distinction is at the heart of my question.

@anongoodnurse, I agree that "traits that favor selection" includes "traits that favor reproduction"—that's my point! But if you read the debate about zebra stripes, biologists aren't content with a circular explanation like "zebra stripes favor reproduction"—they are looking for a specific advantage such as "the stripes help them regulate their body temperature." My question is, why do biologists require such a specific explanation, if examples like mine are possible?

A male just happens to be born with an interest in pattern “s”? Why would that happen in one jump? The possibility of this happening follows directly from the following two premises: 1. Sexual preferences are influenced by genetics. 2. There are random variations in genes. Which of these premises is wrong?

At any rate, the concept of "genetic drift" mentioned by @John seems to best capture what I am asking about here. Let me reword my question to use this term in hopes of reducing confusion.

you are still going to have the issue with this being a very opinion based question. but as a side note something like visual patterning is unlikely to be neutral since vison is how predators and mates notice and identify you.

Sure, and I 3iterate: Zebra stripes are a motivating example for this post, but not the central theme. If you believe that zebra stripes do confer a specific adaptive advantage, then please substitute "zebra stripes" with a trait whose adaptive advantage you cannot identify.

@Max That's not really fair and seems to be a goalpost move. You posit that people search for adaptive explanations for things that need not be adaptive. Your evidence is so far one example; if that example isn't valid, why should we take your premise as true?

I don't know if the premise is true—one possible answer to my question is, "The debate about zebra stripes is unusual. In general, biologists assume that most traits arise from evolutionary drift." Is this your answer? I am asking because I don't know, not because I am trying to checkmate biologists. If I had a comprehensive knowledge of biology, then I would have chosen a better example, but then I wouldn't have needed to ask in the first place.

Ok, I will take on face value that you’re not trying to checkmate biologists. In that case, instead of arguing in comments, why not learn from them? You’re not going to get knowledge from arguing illogical arguments. Genetics doesn’t work the way you want it to work. A mutation is a point in a gene. It’s not likely to result in a completely new behavior. There are exceptions, but they are few and far between, and exceedingly difficult to prove. Lactase persistence is one, and it spread rapidly because of survival advantage. In fact, there are several SNP mutations that have similar results. …

…and we know this because of a lot of time, money, and research in different lactase persistent populations. What would prompt research into your scenario? I suspect nothing would, because it’s not a workable hypothesis.

With all due respect, I resent being accused of "arguing" when I have made every effort to understand the comments here, ask follow-up questions, and use your feedback to improve my post. From these two comments, it seems to me that your view is that "most traits, such as zebra stripes, have a specific adaptive advantage and aren't merely due to genetic drift." Is this an accurate summary? If so, please provide it as an answer rather than a comment.

This article may be an interesting read for you. You can also check out the Wikipedia pages on adaptionism and constructive neutral evolution.

The very shortest answer, and in my opinion most ideologically neutral, is that neutral evolution ("genetic drift") is the null hypothesis of evolution.
The burden of proof is always on the person claiming adaptation. If you can't provide sufficient evidence for adaptation, the default is to assume neutrality.

This is a very surprising answer, if true, given that "open questions" surrounding the adaptive benefit of traits seem to be such a hot topic of research. My impression from skimming the Nature article you linked (but I will take a closer look) is that it is arguing that genetic drift should be treated as the null hypothesis, the subtext being that in practice it often isn't.

@Max "selectionist"/"adaptationist" arguments emphasizing the role of active selection in shaping observed diversity are often dominant, yes. It's more fun/publishable/suitable for textbooks to do work on surprising things than on boring things, even if boring things are more numerous. Hyperdarwinist views are emphasized by evo psych, "new atheism", etc.... To a degree you could call it an overreaction against creationism. For an old but well known criticism, see here.

Is "hyperdarwinist" a synonym or antonym of "selectionist" here?

@Max neither - selectionist/neutralist differences are reasonably opposing views about how traits evolve. "hyperdarwinist" are those who unscientifically try and shoe-horn every observation about the natural world into some kind of story about selection.

@Max - you say you just want to learn, but your arguments don’t support your statement.

In order to gain a deeper understanding of the topic, I ask a lot of "why" questions. I am asking in good faith.


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"Spontaneous Immortalization"
HaCaT keratinocytes are described as "spontaneously immortalized "(see paper below) due to aneuploidy or chromosomal alteration.
From the paper I get that this is a literal term, that the cells just continued growing (?) in vitro and never went into senescence without any "human-induced" transformation.
Is this a common procedure for cell immortalization or a rather rare example?
[http://jcb.rupress.org/content/106/3/761.long][1]

It depends how you define it. Many early cell lines (as well as some modern ones) were derived from tumors. Tumors are spontaneously immortalized cells which became immortalized in vivo. If you're asking how often researchers collect primary cells (non immortalized) and grow them with the hopes they will become immortal the answer is pretty much never.


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Are Oct4 and Oct3 the same protein?
Are the transcription factors Oct4 and Oct3, who are encoded by the POU5F1 gene, actually the same protein, or alternative spliced products from said gene?

The best review I can find is the one by Wang and Dai 2010, conveniently titled starting with "Concise review".
From the beginning of the abstract:

The human OCT4 gene can generate at least three transcripts (OCT4A, OCT4B, and OCT4B1) and four protein isoforms (OCT4A, OCT4B‐190, OCT4B‐265, and OCT4B‐164) by alternative splicing and alternative translation initiation

and from later on, in the introduction:

It is well established that OCT4 gene (official symbol POU5F1, also known as OCT3, OCT3/4, OTF3, and OTF4) functions as a master regulator in maintaining the properties of pluripotency and self‐renewal of ES cells

I take from this review that OCT4, OCT3, OCT3/4, whatever you want to call it, all refer to the same gene. However, this gene does have splice and translation variants. It seems that when talking about these variants, you will see them differentiated using A/B/B1. An older paper and some that cite it, Takeda et al, 1992, refers to the splice variants as OCT-3A and OCT-3B. If not noted, you can expect that the paper is considering them all together regardless of whether they call it OCT3 or OCT4.
I did find one thesis that treats Oct3 and Oct4 as separate proteins, renaming them from the A/B terminology, but they cite only the Takeda paper for this, which does not do the same. It seems more likely to me to be a mistake.

Takeda, J., Seino, S., & Bell, G. I. (1992). Human Oct3 gene family: cDNA sequences, alternative splicing, gene organization, chromosomal location, and expression at low levels in adult tissues. Nucleic acids research, 20(17), 4613-4620.
Wang, X., & Dai, J. (2010). Concise review: isoforms of OCT4 contribute to the confusing diversity in stem cell biology. Stem cells, 28(5), 885-893.

Seems like they are the same. I found the below by using the search terms of "genbank oct3"
I see the names and splicing variations at:
"Atlas of Genetics and Cytogenetics in Oncology and Haematology"
http://atlasgeneticsoncology.org/Genes/GC_POU5F1.html
and also see a statement calling it oct3/4
at:
https://www.spandidos-publications.com/ijo/52/6/2069
which is "expression of Oct3/4 in human breast cancer and normal tissues"
Authors: Feng-Qi Zhao Yogi Misra Da-Biao Li Marilyn P. Wadsworth David Krag Donald Weaver Joseph Tessitore Da-Wei Li Guo Zhang Qing Tian Katie Buss
View Affiliations
Published online on: March 29, 2018     https://doi.org/10.3892/ijo.2018.4341
Pages: 2069-2078


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Unknown Lamiaceae plant identification
A while ago I received a pack of seeds described as Pycnanthemum Pilosum / Mountain mint. The seeds are small (below 1 millimeter) and brown:

Plants grown out of these seeds are around 40 centimeters high and spread widely. Leaves are bright green and smooth, around 3 - 4 centimeters long:

with strong minty smell (though quite distinct from let's say Mentha × rotundifolia or Mentha spicata).
Stems are square, with very short hairs.
Recently plants started to flower:

producing clusters (around 1.5 cm in diameter) of small (3 - 4mm long), slightly  flowers along the stem. Flowers are white, with light shade of purple. with no spots or other marks.
The plant started to flower in the first year after sowing (the plant is around 4 months old, planted outside, in Central Europe).
The plant doesn't really resemble Pycnanthemum pilosum descriptions I found online (for example this or this), which, among other differences, describe long and narrow leaves.
I wonder if someone can help me identify it. Thanks in advance.

I guess I'd like to know which characters don't seem to match.

@BryanHanson Not sure if I get your comment. Could you elaborate? Thanks in advance.

Your edit helped.  So, chief discrepancies are the leaves are too broad, and, it's not hairy. I looked up some pictures a colleague of mine took here in Indiana, which we ID'd as P. pilosum and it's definitely not your plant.  Given the similarities of many mints I think one has to start fresh and attempt an identification.

Can you say more about the flowers? Size? Color?  Spots?  In the one pic they look pretty pale off-white.  Any odor to crushed leaves?

@BryanHanson I've added another picture of the flowers - they become more prominent since I've posted the question (sorry for the quality, I have access only to a phone camera, and it is not great. I'll try to take another one, when the lighting is better). There are small, whitish with a shade of pink / purple, and the color is uniform (including stamen).

That's a pretty great picture actually, and the flower color rules out my answer below.  Do the leaves smell strongly minty?

@BryanHanson They do, which makes me think (following the source you've shared in your answer), that it might be Mentha arvensis.

That's where I was headed if you answered with minty smell.  Will revise my answer for posterity.

I'm going to take a stab at this. If your plant is not particularly aromatic, I feel pretty confident it is Lycopus uniflorus.  There are good descriptions here and here.  In particular, the description of the flowers at the Minnesota site, as having 2 purple-stemmed exserted stamens seems to match your flowers.  Apparently this species should have a tuber (should you end up digging yours up), while Lycopus virginicus which is probably a good second guess does not have tubers (see the differential description at the bottom of the Minnesota page).
Updated and Revised Answer
With additional pictures of now more mature flowers, which are clearly lilac in color, and the info that the leaves are strongly minty smelling, we'll revise the answer to Mentha arvensis, which fits all the available info. More here.


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What species of wasp is this? And how to get rid of them?

Yesterday while moving a chair in my garden I got stung by a group of wasps. During the night I went hunting for their nests and managed to neutralize 3 of them: one was in the chair's legs, about the size of a child's fist and with a dozen on them inside. It was made of grey hexagonal cells, similar to paper in appearance.

(Sorry for the blurry image, I was a bit scared :-P)
Then I've found another one in a water hose tube that was slightly smaller, but quite active.
And finally a third one in a vase, it was very small, only had a couple of cells in it and wasps were working on it, almost as if they were building it from scratch.
Given the number and size of the nests I'm afraid that they could be spreading in the garden. I have already noticed that a couple of them survived the massacre of last night and I'm worried that unless I kill every of them they might keep nesting.
I don't know much about the hierachical society of wasps, but I was not able to identify the queen (although I haven't checked every single one I've killed). Reading online I've found that killing the queen might mean that the worker would eventually die.
So what kind of wasp is this? What can I do to make sure that they do not keep bothering me?
EDIT: by the way, I live in northern Italy

For species identification questions, you should also mention the geographic location of the object in the picture.

Long hind legs, two yellow spots on the otherwise striped bum... I would just ignore these, for two reasons: First, depending on where you live it may cost you a lot of money if you are caught killing them, and second, this particular type is quite harmless and non-aggressive, and most importantly, they feed on mosquitos. My enemy's enemy is my friend, so...

@Damon I wouldn't say harmless and non-aggressive. Just by moving that chair around I got stung and it was quite painful

Where is your chair and garden located?

These are European paper wasps.
(They look very similar to yellowjacket wasps but you can notice the antennae; they are brown here whereas they are black in case of yellowjackets. There are other subtle differences too.)
See this post on Gardening & Landscaping stackexchange about getting rid of wasps.
(This part is probably not really on-topic here and I don't want to copy-paste an existing answer in another stackexchange site.)

If you are upvoting this post then please also upvote the original one in G&L SE

Do European paper wasps count as "dangerous" for the purpose of the accepted answer on the question you linked to?

@T.E.D. I haven't faced them myself. In general wasps are more aggressive than bees. This one is less aggressive than yellowjackets.

Stay vigilant. As you've noticed, once wasps get established in an area, they'll reproduce and spread out. Continue to watch for and eradicate nests. Hardware stores will sell a variety of chemical sprays, but a mix of liquid soap (dish detergent) and water works just as well.
Use wasp traps to prevent wasps from establishing new nests in your area. You can buy them from most stores or make them yourself using directions similar to those provided here: https://www.bobvila.com/articles/homemade-wasp-trap/.

To keep these pests away from your house and other areas, on the underside facing up use a sky blue paint, along soffits, including the bottom of some patio furniture. When these see the sky blue they think its the sky and will go some place else to build a nest. It's a common practice down in the South such as NOLA or other Southern places.


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Any idea what plant is this?

I was asked by my biology teacher to find out it’s name but it seems like a pretty common plant to me so I have no idea how to start. Any idea what type of plant it is?
It was found in Malaysia in my school compound

If you know where and when this was found, this could be helpful extra information to add to your question.

I have no idea how to start

You probably need an "identification key", or some kind of guide, that will help you identify the plant based on a series of questions or identification criteria.
To be able to use such keys or guides, you will need to familiarize yourself with technical vocabulary used in plant description. Some idea of the kind of ecosystem where the plant lives in would likely help too.
If the plant seems common, you will maybe find it on listings of common plants for your area.

This branch of the plant look very similar to the Indian Curry Leaf plant -Murraya koenigii.
It is used as a spice, garnish and also as a taste enhancer in Indian cuisine. It grows in almost every Indian household in their gardens or kitchen for fresh use.

It could be some thing else too, I am a little bit unsure because the edges of the leaves look rough but in Indian Curry Plant, The leaf edges are smooth, Here are some pictures...

I hope this helps in guessing the species,
Best Wishes!

Leaf margins, as you say, do not match. Your answer also lacks citations and does not rectify why this Indian plant would be in Malaysia. Please avoid simply guessing and only answer if you can provide adequate support. Thanks

The Leaf Edges look rough, I agree to that but that could be due to the drying of the leaves for storage, also the Curry tree as it is called, is also found in Malayasia, as the growing conditions required for the plant as same for both the regions (India and Malayasia). Please avoid simply commenting by looking into the answer superficially.

but that's exactly my point. Your answer needs to be complete and stand on its own. The points I made in my previous comment are serious concerns when determining the validity of your ID. Such concerns should be met with cited support if you'd like to receive positive voting response here. Please see [help] and here and here for further guidance. Thanks!


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Clarify pre-column pressure vs. system pressure vs. backpressure for prepacked columns used with the äkta purification system
Background: I am using the Äkta pure system for protein purification, with prepacked columns from GE-healthcare. Just to take one example, I use the HisTrap 5 mL column (IMAC: immobilized metal affinity chromatography). I understands the effects of over-exceeding the pressure limits of a column, and therefore vigorously set the pre-column pressure to 0.5MPa everytime I use the äkta. However, I am starting to question or how I should think about the different pressure, and if 0.5MPa is correct for the different columns I am using.
I was taught to always set the pre-columns pressure, which makes sense since as far as I can tell – this is the actual pressure inside the column (pressure felt by the resin). However, If I open the documentation on the HisTrap 5 mL column from GE-healthcare, I can’t seem to find much about the pre-column pressure limit. I also find the terms max-backpressure limit in the documentation for the column, and system pressure.
Questions: How is the system pressure different from the pre-columns pressure? Should I calculate the pre-column pressure myself as an effect of the size and length of the tubing leading to the column? Is there a good article or webpage that goes through this theory at a basic level?
It seems obvious, but how can backpressure be created in a system that is not closed and goes one way (or perhaps the Äkta is creating the pressure)?

https://www.google.de/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0ahUKEwirg8XYmZTZAhUEiSwKHTUOC_MQFggoMAA&url=http%3A%2F%2Fproteins.gelifesciences.com%2F~%2Fmedia%2Fprotein-purification-ib%2Fdocuments%2Fhandbooks%2Fakta-chromatography-systems.pdf%3Fla%3Den&usg=AOvVaw2v8INehaa7HnPDoEEtp8Dk Chapter 7 of this document may be of help.

Is there a good article or webpage that goes through this theory at a basic level?
Yes, Chapter 7 of the manual linked to by Andreas Tosstorff is a good source, and is also the source of the images below. The manuals for your ÄKTA system and the HisTrap columns discuss pressure in some detail as well.
How can backpressure be created in a system that is not closed and goes one way?
This is a physics question and, not being a physicist, I can't go into detail. However, I also use the ÄKTA system for FPLC and may be able to provide some form of an answer. Any fluid moving through tubing experiences some resistance from, for example, friction between the fluid and the tubing walls, bends in the tubing and obstructions like filters, mixers, valves, etc. Even though the system is open at one end, there is resistance to the flow of fluid through it which creates backpressure. The largest sources of backpressure in the FPLC system at low flow rates are filters, flow restrictors (if installed) and the column itself.
How is the system pressure different from the pre-columns pressure?
The system pressure for the ÄKTA is measured at the pump. This measurement in itself is not very informative. The pressures that are important as far as keeping your column safe are the pre- and post-column pressures (the pressure measured immediately before and after the column, respectively), and the difference between these two (the pressure across the column). Your column has two pressure limits:

Hardware pressure limit: this is the maximum pressure the column itself will withstand before there is a risk of failure. I know this from experience after a malfunctioning valve gave 4 times the maximum hardware pressure to my column. In my case, failure meant the top of the column blowing. The pre-column pressure should not exceed the hardware pressure limit.
Resin pressure limit: this is the maximum pressure the resin within the column can withstand before collapsing. The pressure across the column should not exceed the resin pressure limit.

Should I calculate the pre-column pressure myself as an effect of the size and length of the tubing leading to the column?
Some ÄKTA systems are capable of measuring pre- and post-column pressures directly. Mine is not, and I don't know about yours. In the case where pressure is measured at the pump, the pre-column pressure is equal to backpressure between the pump and the column (caused by the tubing/filter/etc.) subtracted from the measured system pressure. To determine this, you essentially disconnect the top of the column, run the system under your intended conditions and record the measured pressure (this is the backpressure generated before the column). The manual for my columns (HiTrap Q) explains this step by step, and yours may as well.

The manual also explains how to determine pressure across the column in a similar manner. However, in my experience, the flow path after the column is relatively unrestrictive (unless a flow restrictor is installed), and its contribution to backpressure is relatively be negligible. Thus the pressure across the column is reasonably equal to the pre-column pressure, which is also reasonably equal to the measured system pressure. Furthermore, listed in every manual I read is some statement to the effect of:

The maximum pressure over the packed bed (Δp) is an approximate limit. It is dependent on the characteristics of the chromatography medium and on sample/liquid viscosity. The measured value also includes the pressure generated by the column tubing. For many columns, the recommended flow rate is a better guideline for protecting the packed bed.

In fact, the manual for my columns doesn't even list the resin pressure limit and it took substantial digging to find out what it actually is. I always run at the recommended flow-rate and set the system pressure alarm at the resin pressure limit (0.3 mPa in my case versus 0.5 mPa hardware limit) without any of the corrections mentioned above. However, depending specifically on your system and usage, it may be necessary to make them.

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When in Ampicillin degraded (gone) in liquid TB-media? Concerns about selectivity
Question: Specifically regarding Ampicillin; When growing cells in TB (terrific broth) for protein expression - when should I expect the ampicillin to be gone due to degradation by b-lactamases? (and how much Ampicillin do people add in addition to standard 100 ug/ml)
Background: I have heard rumours that all Ampicillin (100 ug/mL) is degraded within few hours (2-3) due to the enormous amount of b-lactamases secreted by the bacteria.I have also heard that some are not concerned about this, because 2-3 hours is anyway enough for the culture to outcompete anything else that will grow after the ampicillin is gone. On the other hand, some people are also writing that they add more ampicillin every 2-3 hours to maintain selectivity; however, I dont really see the point in maintaining selectivity as long as I have plenty of protein I will anyway lyse the cells and not use them for anything else.
My growth conditions: Grow at 37 degrees till OD600=1 (takes around 3h), then induce with IPTG and grow at 25 degrees for 18h, 200 rpm the entire time.
End comment: I know you can generally change to the more expensive carbenicillin which is degraded slower, but i'm more interested in knowing "how bad" Ampicillin really is for my general knowledge (and I will anyway keep on using it). Also: Any general thoughts or references to literature on this topic, specifically for Ampicillin is appreciated.

rather interesting question. Selection pressure is very important for any type of work. And it is true, amplicilin will degrade in media due to the secretion of beta-lactumase. This is a limitation that we must face, though there are ways of getting around this.
For example, when cloning, on a plate sometime you will notice "smaller" colonies growing around a central large one. This is due to beta-lactumases. They are not a problem if you are going to take the central colony and place it into fresh media with new amplicilin. They will simply die out. No harm done.
In liquid culture it is good to not "oversaturate the culture". Usually, do not let it get over OD600 = 1. In addition, do not use old stocks, as this causes problems with selection and use higher concentrations of ampicillin if you are still having troubles selecting!
Generally speaking, 100ug/ml is standard for most selection with cloning, and should be sufficient for what you are doing. It seems that your protocol should work fine for protein isolation.
Last remarks -- it is a problem that most microbiologist do not think of. Ampicillin has its limits!
https://www.addgene.org/mol-bio-reference/antibiotics/
https://bitesizebio.com/10188/whats-the-problem-with-ampicillin-selection/

Thanks, although this is all true - is there still an answers to how bad ampicillin is? Is it really gone from the media within 2-3 hours? Also: when using TB you really should not expressed before the OD600 reaches over 1, because IPTG will not be taken into the cell before the glycerol is used up (TB can theoretically support growth up to OD600=16)

Also, are you saying that the Ampicillin is likely gone after OD600=1?


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Does glycerol in E.coli culture media somehow inhibit the lac-operon?
I have have been taught that one should induce protein expression with IPTG at an OD of about 1.0 - 2.0 when E.coli grows it TB media (terrific broth). As a reference point, one typically induces protein expression at an OD of about 0.5 in the commonly used LB media (Lysogeny/Miller broth). IPTG is a lactose analogue that will induce the expression of proteins encoded in the lac-operon. I have been told that it is important to have a high OD before induction, because the added glycerol in the TB media (5%) will somehow inhibit expression of protein in the lac operon if it is not 'used up' before induction. However, glycerol itself should not inhibit the lac-operon from being transcribed (?).
I have several speculations about this: Could it simply be that TB-media has a very long exponential phase (rumoured to support an OD up towards 16.0, personally I routinely reached an OD of 10), and it is therefore not wise to induce protein expression too early (and thus has nothing to do with using up up the glycerol)? Another rational explanation that I came across is that glycerol might induce carbon catabolite repression, meaning that glycerol might inhibit the uptake of IPTG (or down regulate cAMP levels) and thus indirectly inhibit expression of protein from the lac-operon, but I found it a bit difficult to get a straight answer about this in literature.
Does anyone have some good references for these speculations?

You can induce as soon as cells go into exponential phase. It is during the lag phase where you should avoid burdening the cells.

Yes, I was however wondering about the use of glycerol and TB media, i.e. why 'everyone' says to wait for an OD of 1 or more before induction? Are you saying that this is a myth, and it simply does not matter as long as one is in the exponential phase? Or that it simply takes longer to get to the exponential phase in TB media, and thus the recommendation of inducing at a higher OD? I actually haven't document the growth curve in TB-media, I have simply used or modified protokolls, which has worked well for me.

I cannot provide a conclusive answer here but how soon you induce depends on the load of protein expression. It would vary for different kinds of media. I haven't used TB media, so I can't say much about it. A long-lived protein will reach 90% of steady state concentration within 4 divisions. Based on these factors, you can decide when to do the induction to minimize stress and maximize protein concentration.


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Why is sorbitol used in buffers?
Many protocols in my lab use sorbitol in buffers.  For instance, in co-immunoprecipitation, we include it at a final concentration of 200 mM in our lysis buffer. I'm not entirely sure why though.  I believe it might be as a crowding agent. But even if this is the reason, I'm not entirely sure what this means.  Could someone explain?

Generally, sorbitol is one of those 'old tricks' in the book which is thrown into cultures or buffers for several reasons. Here are a few that I have heard of:

Bacterial cultures (e.g. E.coli BL21) that are optimised to express (produce) protein, because sorbitol is thought to increase the expression of soluble protein. I have even heard of people (who work with very difficult proteins) that add to a final of 0.6M sorbitol in their culture media (note that the cultures grow very slowly).
In buffers intended for proteins, as it is thought to increase the solubility (i.e. avoid aggregation), similar to other sugar such as glycerol or osmolytes such as betaine.
Sorbitol is considered (chemically)  to be relatively inert, and many will therefore prefer to add sorbitol over e.g. glycerol or sucrose as a stabalizating agens when they work with proteins.
When you work with glycoproteins, you are generally depending on the addition of some sugar to stabilise the protein of interest - both during isolation, purification and analysis/testing.

The last point here could answer you question, as immunoglobulins are glycoproteins, you are generally depending on a high concentration of some sugar - such as sorbitol - to work with these proteins (i.e. antigens and antibodies) in e.g. immunoprecipitation.
I don't believe that sorbitol is a crowding agent - as this would perhaps facilitate aggregation, the opposite of the intended purpose. Adding crowding agents it typically done in protein crystallography with the addition of various PEGs to 'get the' proteins to form crystals. Instead, I think that sorbitol may (somehow) interfere with the proteins solvation shell - in the same way that is speculated for osmolytes (e.g. betaine or various sugars); however, this is something that I dont know much about and should be investigated.


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Why are some protein sequences known but their 3D structure isn't?
Why are there some proteins that have a known amino acid sequence, but their 3D structure is not known? Wouldn't finding the former in a lab lead to the discovery of the latter? Please correct me if I have misunderstood something.

related https://biology.stackexchange.com/questions/85723/why-does-it-matter-to-predict-protein-structure

Protein sequencing is a nicely constrained problem: you have a one-dimensional sequence of amino acid members, which come from a limited set of options (made a bit more complicated by post-translational modifications, but not much more so). Because it's one-dimensional, it's a problem you can easily solve by chopping up a protein into little bits, using mass differences between amino acids to understand their constituents, and determining the order from that distribution. If a DNA (or mRNA) sequence is known, it becomes even easier - you can skip the protein sequencing process and get the amino acid sequence directly from the nucleic acid sequence and the genetic code.
By comparison, protein folding is an absolute nightmare to solve for. Chemical bonds between amino acids are not rigid, they can bend and twist in all directions. The conformation of those bonds also depends not just on adjacent amino acids (as in a 1-D problem) but potentially on any other amino acid in the sequence (not to mention external influences..).
In a large molecule like a protein there is a massive massive degrees of freedom problem. From Wikipedia, describing Levinthal's paradox, bold mine:

In 1969, Cyrus Levinthal noted that, because of the very large number of degrees of freedom in an unfolded polypeptide chain, the molecule has an astronomical number of possible conformations. An estimate of 3300 or 10143 was made in one of his papers[1] (often incorrectly cited as the 1968 paper[2]). For example, a polypeptide of 100 residues will have 99 peptide bonds, and therefore 198 different phi and psi bond angles. If each of these bond angles can be in one of three stable conformations, the protein may misfold into a maximum of 3198 different conformations (including any possible folding redundancy). Therefore, if a protein were to attain its correctly folded configuration by sequentially sampling all the possible conformations, it would require a time longer than the age of the universe to arrive at its correct native conformation.

Now, of course that's not the actual process that proteins use to fold (they don't iterate through all possible combinations, they settle through an energy landscape where only certain intermediate conformations are realized), and we can use that in computational models to solve protein structures more quickly than the age of the universe, but it's still quite a slow process. Projects like Folding@home have aimed to distribute the computational load among unused processing power in devices around the world, including idle gaming consoles and personal computers, but there are many many protein structures to solve.
It's possible to get a general picture of protein shape using imaging techniques like X-ray crystallography or cryo-EM, and for some purposes these techniques give a lot of information, but these techniques are also by no means simple and can be prone to errors.

Furthermore, the methods used to experimentally determine protein structure (e.g. x-ray https://en.wikipedia.org/wiki/X-ray_crystallography and cryo-EM https://en.wikipedia.org/wiki/Single_particle_analysis) are not one-size-fits-all solutions and are move effort-intensive than protein sequencing even in the best of cases.

You also don’t necessarily need to sequence a protein if you know its gene sequence.

@canadianer I kind of mentioned that, maybe I can make it more clear...

You did. I’m sorry I missed it.

I think you get more than a general idea of the structure from x-ray crystallography (!?), depending on resolution of course, I would even dare to say that you cannot possibly get more structural information anywhere else than you would from a high resolution crystal structure. Which is why some researchers are specialized x-ray crystallographers). CryoEM is also catching up, and NMR studies are good form small proteins and dynamics.

In my opinion, the focus on protein sequencing here is a bit 'pre-DNA-area', as we today nearly always do recombinant expression with protein sequences from DNA (i.e... we rarely isolate unknown proteins from natural samples and sequence them by chopping up the protein by hydrolysis).

To answer why sequences are known before structures, it is worth highlighting the typical ‘workflow’ for a biochemical researcher. Briefly, sequence is always before structure because you need the sequence to determine the structure. As with everything else one would like to investigate, you have to start with the information that you already have. In modern sciences these are usually as following for protein research:
Workflow:
1. Isolate some bacteria or fungi from e.g. the ocean or anywhere else, and sequence their whole genome (DNA). This is very realistic to do, and not that expensive anymore.
2. Once you have the genome sequence, there is a lot of bioinformatic work to be done to annotate the sequence. This means, identify coding regions for e.g. for proteins. There are programs that are very good at this, because we already have a lot of information on what is encoded in living organisms (based on experimental data and years of research).
3. The DNA annotation programs can assign thousands of proteins in one genome. These protein sequences are then uploaded in relevant databases, for other people to view and work with. Note that these proteins sequences are NOT experimentally validated. They are however assumed to be correct with some statistical validity (which is usually correct now a days due to the overwhelming amounts of collected knowledge, and sophisticated software).
4. Scientists (i.e. biochemist and others) can then work with these protein sequences to find out if they actually are what the programs assumes. This involves the bottleneck of actually been able to produce and purify the protein of interest (which may be very difficult).
5. After experimentally assuring that the protein actually has the function you are interested in (by doing experiments), and being able to actually produce and purify it, one would typically want to determine its structure. This is because the three-dimensional structure of a protein can explain how and why it work the way it does. This is however difficult to do, experimentally (as well described by the other post answer).
Can you trust homology-models?
As a scientist working with protein structure and function, I would also note that (in my opinion) you cannot completely trust structures solved purely computationally (i.e. homology-models). These estimated homology-models are simply based on actually structures that are experimentally validated (e.g. crystallographic). Even though homology-models are very useful when you do not have a better structure, you can never be completely sure that they are correct (as they are simply assumed models of structural models; i.e. models of a models).
The active site of enzymes is of great interest to understand how enzymes catalytic their reaction. It is of vital importance to know how the catalytic residues are structurally placed/arranged in the active site to understand and even modify their catalytic behaviour. Even if you have a homology-model is 98% correct, the 2% error could be the structural placement of catalytically important residues. You also can not know for sure what the error is. One should therefore be very careful to put to much reasoning into a homology-model. In summary, if you don’t have a experimentally validated structure (which is difficult to get) you can never be completely sure of what is going on (or at least, you would be working in the dark - looking for effects).
Experimentally validated structures:
I would also like to add that x-ray crystal structures are, as of today, the golden standard when it comes to protein structure information (although cryoEM is catching up(!), and NMR structure give a lot of information about dynamics). You should check out the PDB database. If you have a high resolution structure, of e.g. 1.1A you are approaching atomic resolution and can even see the rings in the aromatic amino acid side chains (which is very cool!).
To answer your question in brief:
Sequence is always before stucture as you cannot experimentally determine structure without the sequence (also required to computationally model the structure). The protein sequences are simply assigned with complexed programs, from the DNA sequences. After you have the sequence you need to experimentally validate that the assigned protein sequences are correct. Only after all this work ca you start to determine its three-dimensional structure.....through a lot of hard work.


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Insulin and leptin action on Anorexigenic Neurons in Hypothalamus?
Receptors for Insulin are present on liver, adipose tissue and muscles. In addition, there are Insulin Receptors present in arcuate nucleus of hypothalamus, which influences anorexogenic neurons through IRS2 and PI-3K signaling. They also causes the inhibition of Food intake by increasing the expression of POMC, and cleaving its protein to alpha melanocortio stimulating hormone (αMSH). This is however the same response produced by the release of "LEPTIN" that act on anorexogenic neurons, and stop eating behavior - similarly as insulin by producing (αMSH).
My question is: why insulin goes to brain and produce this effect when their was Leptin available to do the same task? What is the significance of Insulin action here?

This question is identical in nature if not in subject to this one. I'm a bit puzzled. Can the body not have several pathways for achieving a "goal"? Why, to you, should this overlap not exist, since clearly it does, and we seem to be surviving in spite (or because) of it?

receptors for insulin are present in many more tissues than just liver, adipose and muscle... the insulin receptor is expressed in numerous tissues throughout the body (as well as the leptin receptor - it's not just expressed in the CNS or adipose tissue)... different tissues may express the same receptor but the tissue effect can be very different (as well as overlapping with other hormones).

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gene transformation from plant to plant - transgenic plant
I want to know if the expression of a transfered gene depends on source of the gene, if e.g.:
1] I isolate a gene from plant and transfered it to the same plant?
2] I isolate a gene that is highly expressed in plant, and transfer it to another plant ?
3] I isolate a gene that is lowly expressed in plant and transfered it to another plant
Is there a difference between the expression of the transferred gene in these three cases? What does the expression of the transfered gene depend on?

There are many factors that could influence in the level of expression of a gene, but the two most important are the promoter and the place where your gene has introduce in the genome of de recevier plant.The first one, the promoter depends of the original expression of the original plant, if the expression was high in the original plant, it will be in the receivier plant (if you include the promoter in the construction of the transferred gene and both plant are close in filogenetically).The second one,the place where your gene has introduce in the genome of de recevier plant is a matter of luck, if you use a Agrobacterium, if you gene was introduce in a heterochromatin region it won´t express.
Sorry for my bad english, hope I was useful


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Cold adaptation in proteins, at sequence & structural level
When looking for cold adaptation in unicellular eukaryotes there is not much work found. Most of the time general sequence comparative sequence analysis between marine mesophile & psychrophile also does not give clear results. If I am planning to study at structural level (homology modeling) will I get answers? What type of parameters can I look to study? Which proteins are expected to provide cold adaptation apart from ice binding or antifreeze proteins because IBP & AFP cant be found by sequence comparison. Is it possible to give some ideas regarding this?
Thanks

There are research groups working on this exact question; understanding cold adaptation in enzymes. You can read about this in several articles such as 'Computation of enzyme cold adaptation', or 'Molecular Structural Basis for the Cold Adaptedness of the Psychrophilic β-Glucosidase BglU in Micrococcus antarcticus', or 'Specific amino acids responsible for the cold adaptedness of Micrococcus antarcticus β-glucosidase BglU'
On a general level there are several sructural characteristics that are typically found. They are usually far more flexible (i.e. less salt-bridges and other such stabilising bonds). The flexibility come from having fewer strong bond between and within the polypeptide chain, and even containing disordered regions. The flexibility also means that they are usually far less stable, and more difficult to work with. They are industrially interesting, as developing enzymes that can work at low temperatures can reduce the cost of running reactions. This is essentially a step towards a greener-industry. There are also speculations about various ratios and abundance of the amino acid types, and where they are located.  You would have to read articles on specific proteins to investigate this.
From a practical view-point, cold adapted enzymes are usually isolated from arctic microorganisms and characterised in the lab - in order to determine their temperature tolerance. The structure of such enzymes are then solved (after lots of work) and form the basis for understanding cold-adaptation.
To answer the question more specifically, no its not realistic to know if something is coldadapted directly by looking at the protein sequence (on its own), this has to be determined experimentally in the lab. However, if the protein is from a cold adapted organism (typically arctic bacteria) there is reasons to assume it is cold adapted before you test it in the lab. You can also apply som bioinformatics (e.g. blast search) to determine if its related to cold adapted proteins.  Also, If that specific type of protein that you are looking at, is well studies, you could also try to make homology models and see if its structurally similar to cold-adapted relatives or if it is more structurally similar to its mesophilic or even thermophilic counterparts. This is however very speculative, and i would not put too much weight on theoretically assumed adaptations - you should at minimum know that the protein of interest is from a cold adapted organisms, to even begin to look (computationally) on why and how it could be cold adapted.


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Enzyme kinetics; what happens at the peak of the Gibbs energy graph?

At the very peak, the energy is in a state of activation energy. Here, is the substrate just attaching to the enzyme, or is is substrate already breaking?

Look up transition state.

Enzymes are catalysts that lower a reactions activation energy (Ea), and thus increases the rate of the reaction. The diagram above is a Gibbs-Free energy landscape/pathway, where the top of the diagram is the transition state between the substrate and the product. Enzymes speed up chemical reactions by stabilising the transition state, and in effect, lowers the activation energy (Ea) required to transform the substrate into the product. The active site of enzymes are thus specifically 'designed' to be as energetically favourable as possible for the transition state.

To understand enzyme kinetics it could also be a good idea to look at (understand on a general level) the Arrhenius equation and reaction rate constants (k), to more completely understand what we mean by activation energy and reactions rate.
This whole topic is taught in basic biochemistry, and thus well described by many educational websites, all biochemistry text books, and even found in  educational youtube lectures (which is perfect for a basic understanding before you read more heavy theory). My earlier biology SE question Enzyme kinetics: recommended literature to grasp the concepts better also has some good book recommendations.
Following are also some webpages you can look at.
Khan Academy:

With the catalyst, the activation energy is lower than without. ...
That's because enzymes don't affect the free energy of the reactants
or products. Instead, enzymes lower the energy of the transition
state, an unstable state that products must pass through in order to
become reactant (...)

Chemistry LibreTexts: General transition state stabilisation is also a nice keyword that would give you a lot of information, e.g. from Chemistry LibreTexts:

Linus Pauling postulated long ago that the only thing that a catalyst
must do is bind the transition state more tightly than the substrate.
That this must be the case can be seen from the diagram (...)

Wikipedia: It could also be a good idea to generally read about catalysts, here from the wikipedia page where they use the classical example chymotrypsin and its oxyanion hole (active site that binds the substrate) that contains the Asp-His-Ser catalytic triad:

In the presence of chymotrypsin, (...), a nucleophile is used in the
form of the catalytic triad - Asp 102, His 57, Ser 195 side chains.
Moreover, the oxyanion hole, which consists of the backbone -NH-
groups of Gly 193 and Ser 195 of the enzyme, have the N-H groups
positioned in such a way that they will donate strong hydrogen bonds
to the substrate's C=O oxygen, given that the carbon atom is
tetrahedral as found in the transition state. This strains the bonds
of the trigonal planar C=O of the original substrate, helping the
reaction to proceed to the transition state. The hydrogen bonds also
stabilize the formal negative charge on the oxygen atoms. In this way,
the activation energy of the reaction is lowered and the rate of
reaction thus increases.


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Woodpecker pecking, an effective evolution?
I was sitting out on my balcony earlier and saw a woodpecker land on a tree nearby. It began pecking at the tree, and did so about five times, in intervals of about four pecks each time, before finally giving up and flying away. My question is quite simple, were woodpeckers gifted(did probability glean favorably upon) with a positive form of evolution? Does the reward justify the amount of energy they have to exert for food? Were they given an improper and doomed form of evolution?

Welcome to Biology.SE! What do you mean by "were woodpeckers gifted with a positive form of evolution?" and "Were they given a improper and doomed form of evolution?". Do you mean "Is the pecking behaviour of woodpeckers adaptive or should they better do something else of their lives?" Out of curiosity, do you know which species you were looking at? (it might not matter much)

@Remi.b "should they do something else better with their time". And no, I could not see it for it was partially a silhouette, it just looked like a traditional woodpecker to me. I live in Kansas city near the border of Missouri if that matters at all.

I mean, if pecking trees didn't help woodpeckers survive, they wouldn't do it. They're specialized to exploit the food niche of bugs in trees. The reward does (biologically) have to justify the energy they exert for food, or else the woodpecker would die.

The question does not deserve downvotes. I upvote in reaction to the downvotes.

@Luigi: I would look at it the other way around.  If they didn't get enough food energy from pecking trees (and eating the bugs they find there), they would have starved to death long ago, and there would be no woodpeckers around.  The fact of their existence demonstrates that they can survive.

As a +1 to @Remi.b , would downvoters care to explain why this question is bad? I cannot see why this question is in any way lacking in research effort, at most it is slightly unclear.

@MarchHo Unjustified downvotes is kind of a regular thing when it comes to Stackexchange and its off-branches. In my opinion, downvotes should require a comment, but that's just me.

@MarchHo I haven't downvoted but can imagine that downvoters feel that the Q lacks research effort (many misconceptions of the evolutionary process) and is unclear, i.e. 2 out of 3 of "This question does not show any research effort; it is unclear and not useful". These problems have also been mentioned in earlier comments, so further comments pointing this out are redundant.

@jamesqf I think you and I are making the exact same point. The first sentence of my comment maybe should read: "I mean, if pecking trees didn't help woodpeckers survive, they wouldn't do it, because they would already be dead."

@Luigi: Yes, we're saying the same thing, just looking at it from different directions.  As with so many other evolutionary questions, there's an inherent circularity: the reason X evolved to do Y is because it did, and if it's around & reproducing it's successful.

My question is quite simple, were woodpeckers gifted with a positive form of evolution?

Though this has been addressed in comments, it's an opportunity to address a popular mind frame about evolution, that is anthropormorphizing it.
Evolution does not think or bestow in the sense that a philanthropist might. Evolution does not doom or treat unfairly as a dictator might. Evolution is merely the process of predominantly random changes in genetic material that result in a change in the expression of that genetic material. If it is a change that radically affects expression of a critical gene (say, that encodes the structure of hemoglobin), the organism experiences a lethal point mutation and ceases to exist. Evolution for that organism stops there; if death occurs before reproduction, the mutation is lost.
Evolution seems beneficial because the environment exerts selective pressures on life: if two animals (say, birds) in the same habitat (say, woodlands) eat the same food (say, larval insects), competition for resources exists, decreasing the numbers of both animals. However, if a mutation (say, a marginally different beak shape) allows even a slight chance that the animal will have a competitive advantage, it is kept in the population. A staggering number of mutations similarly either harmless or advantageous may eventually allow two animals to inhabit the same habitat without competition - a net gain for both animals.
But there's no gift involved. Just a mind-numbing number of non-lethal mutations over a mind-numbing number of years, under the influence of the selective pressures of the environment. You don't see all the mutations that were deleterious, resulting in countless deaths and dead ends because you are standing in one point in time, seeing what exists around you now. The woodpecker seems almost fantastical.

Were [woodpeckers] given an improper and doomed form of evolution?

Evolution follows the same principles for all species: predominantly random mutations without rhyme or reason or form. If you want to speak of doomed animals, look to mankind's effects on the environment in the last 2000 years, or the last 20. When ecological niches are wiped out, that, not evolution, dooms an animal. The Monarch Butterfly is a poignant example of dooming a species by destroying it's habitat.
A great way the understand selective pressures of habitat is to read about marsupial evolution in Australia. The effect of ecological niches on evolution is very clear.

When I say "gifted" I mean "did probability glean favorably upon". And I know how evolution works; my question was more or less about sustainability of the species given their current scope and path of evolution.

@Krythic - it is as sustainable as any species given its current scope and path of evolution. That's evolution. :-)


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Flow cytometry analysis

I've performed an experiment wherein I have stimulated cells (cell line) with a drug across different time-points - in its unmodified (Drug) or modified form (H-Drug or M-Drug).
All cells are seeded at the same concentration and all are harvested for analysis on the same day.
Method:

Cells were seeded on the same day. Cells were stimulated at 3:00 on a Wednesday (24 hour timepoint). Cells were then stimulated at 09:00 am on Thursday (6 hour timepoint) followed by 12:00 pm Thursday (3 hour timepoint) and so on until 3:00 pm on Thursday - at this time ALL cells were harvested and put into separate tubes according to what they were stimulated with and when e.g. 3 hour M-drug.
All tubes were analysed in sequence to measure the percentage of cells in that test tube that stained positive for that drug (i.e. as a means of measuring drug uptake)
Question:
I have measured the percentage uptake of a drug at different time-points (0.5 - 24 hours) for the three drug variants. % uptake is taken as the % of cells that stain positive for that drug
What would the correct stat test be? Would a 3-way ANOVA make sense?

Correct me if I'm wrong but I'd just do one-way ANOVA for 3 treatments. In a 3-way anova you'd get stats for the effect of [a and b over c], [a and c over b], and [b and c over a]. I don't think your experiment is designed to test these parameters in relation to one another, but whether the modification differ from the unmodified drug.


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How to handle glycan structures for bio-/cheminformatics in Python?
I am currently trying to do some machine learning on biological reaction sequences which include glycans. To represent the substrates and products as vectors I am comparing different fingerprints that I calculate using rdkit. For most compounds on found in KEGG this is not a problem as I can download their structure as .mol files and then parse these with rdkit. For glycans this format is not available and I have so far been unable to find any reliable way to turn these glycans into rdkit mols or any other standard structure format like SMILES. Please let me know how I should handle glycans in a chemical/ bioinformatics setting. Ultimately, I just want to calculate a few different fingerprints (Morgan, MACCSKEYS, etc.) and compare. What I need is a a way to read glycan structure files in Python. I have obtained the glycan structures in KCF, WURCS and IUPAC formats but can't figure out how to handle them in Python for further analysis.

If no one answers here, you may have better luck on bioinformatics.stackexchange.com. Be sure to update your question to include a link to the cross-posted question if you do so, or flag your question to be directly migrated.

It would help to know what you mean by "turn these glycans into rdkit mols", what is the starting glycan molecular data format you are looking at? Is it just some label on KEGG or what? See also here.

I have edited the question to add detail on the formats I have available. Thanks for the advice.

The example glycan Sialyl Lewis x antigen from KEGG has a SMILES entry on its on it's PubChem entry. Could you implement a (probably computationally/time expensive) search for the glycan names in your DB and pull the SMILES from the PubChem pages?

I'd try http://bioinformatics.stackexchange.com @MaximilianPress has a good answer.

It seems like there are a few options, I don't know much about these but they will at least allow you to read structures into format that people use for structural analysis:

https://glypy.readthedocs.io/en/latest/Quickstart.html
https://github.com/BojarLab/glycowork

For a particular application see here.


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Can I use yogurt to prevent the growth of black mold?
I have a mold problem in my bathroom.  There is a narrow groove between the top of my tub and the wall it is attached to where a black gunk repeatedly grows.  I can shoot the material out with a spray bottle but it grows back.
I've been taught many times that a bacterial film can prevent the growth of other bacteria, because the film created by one prevents another from colonizing that spot.  I have the inclination to think yogurt has bacteria.  If I were to clear the groove of the material, then spray the groove with a mixture of yogurt and water, would the bacteria in the yogurt colonize the freshly cleared groove and prevent the growth of what I assume to be mold?

I assume this is on topic as I am looking for a sort of corroboration/rejection of this scenario rooted in the biological workings of bacteria.

You need to replace the caulk, use a caulk with a mildew inhibitor.  The applications for what you are talking about would be more for treating vegetables and such like in a garden. Putting yogurt in your bathroom is strange and kinda gross.

@rhill45 - people put yogurt in their mouths and in their bodies? Why would you think a dab of yogurt around the tub is gross?

Because it smells worse than vomit

If it were possible, how would you get around the problem of water often washing away the yogurt?

@rhill45 - Speak for yourself! I would prefer yogurt to vomit any time.

Maybe we should put yogurt in tile, caulk and other hardware products. Sorry I get weary when people think yogurt, vinegar and incense will clean everything in their house. Just a personal pref!

@anongoodnurse I assume if the bacteria in the yogurt could colonize the groove, then it won't matter if the yogurt is eventually washed off, since the little ones will just cling to the surface.

Nice thought, but the bacteria need an appropriate growth medium, which would not exist in that crack area. But, as I said, nice thought. :)

ohhh, so the moist environment wouldn't be enough huh?  Post thy answer, @anongoodnurse

@rhill45 - not to extend discussions in comments, but I would be a happier camper if people cleaned their houses with a lot of water, a little vinegar or baking soda, and elbow grease, leaving all these other silly cleaning products (aerosolized and other noxious, polluting forms) in the warehouses of the money-grubbing, panic creating manufacturers where they belong.

@anongoodnurse good point re safety but if it's a toxic black mold, maybe time to pull out the big guns :/)

You should consider treating the cause instead of the symptoms. Opening the windows can do a huge part in inhibiting mold growth by reducing the humidity. Also, IMO this fits better on Life Hacks or DIY SE.


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Generate T2/T1 Phage Resistant E Coli
I would like to generate T2/T1 phage resistant Stbl3 E Coli to use in virus production. Is there a plasmid somebody has used that confers resistance, or is this done another way?

Two-step resistance by Escherichia coli B to bacteriophage T2 may not give you a specific protocol as to how to accomplish it, but there may be some useful info


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Is there a web tool for species identification?
I know this stack exchange provides the ability to post photographs of animals/plants for species identification, but is there a web resource that can automatically identify a plant/animal using image processing/correlation tools - so the user doesn't have to painstakingly work with keys?

Try searching "species identification website" and there are many websites for species identification. Therefore I have flagged as too broad. Also, you haven't showed much research effort. A simple search gives you so many results.

I recommend using iNaturalist and their mobile app. That is very useful community in species identifications.

Encyclopedia of Life is also a good database for species identification but it does not have "automated tools" to do the search. Any kind of image search would work reasonably well if the training set is big+diverse enough or the query is unambiguous. This is a machine learning problem. Yes the user has to take pains and that is why there are biology courses on these topics.

Scientists are only beginning to develop robust techniques using DNA markers and NIR spectroscopy, for instance. For automated systems based on photographs there are some tests too, but usually restricted to very narrow taxonomic groups.


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Please help me to identify this red worm
I found bunch of red worms coming out of sink.
It is rather unusual to find an overwhelmingly large number of worms. It doesn't seem like earthworm or horsehair worm. I don't know much about the worms though.
Could anyone please identify it for me.?  Notable features are

Dark red in color.
Has  a hook like structure at its rear end.
Moves in both directions front and rear.
It shrinks when it dwells in water.

I am nearly sure it's an earthworm. Location, approximate size and number of annuli (segments) would be of great help in narrowing down the circle of potential species. Also, is it characteristic for them that have no clitellum or is it just a single specimen without it?

Judging from the photo, it's an earthworm, some species of Lumbricidae. If the accident happened in India, this faunistic paper can be of some help.


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Sequencing a specific region of a genome
First off, I'm new to bioinformatics and I am learning about DNA sequencing.
Let's say that I knew that a specific region of a genome which contained information about a disease (whether it a person had the disease or not).
It would make sense that we would only want to sequence that part of the genome in order to make a detector for this disease.
Would it be possible to 'cut-out' this region of the genome and only sequence that part (so we don't have to sequence the entire thing)? If not, how would we sequence only this part of the genome (it doesn't make sense to sequence the other parts as they don't give us information).
Thanks in advance.

Yes, it's possible to sequence a certain region. Probably the most straightforward method would be PCR amplification.

I have been doing a little researching, and I also found something called 'Targeted Resequencing', do you know whether this has anything to do with PCR amplification?

Resequencing is basically sequencing something that has already been sequenced. This means that instead of assembling all of your sequence reads from scratch, you can just align them to the reference sequence (the entire human genome has been sequenced). Targeted resequencing means your sequencing a specific region, such as a gene. The targeting can be done by PCR or by other methods.

Yes, it is possible to sequence a specific region of the genome. The method, as you mentioned, is called targeted sequencing. Resequencing is basically sequencing something that has already been sequenced. This means that instead of assembling all of your sequence reads from scratch, you can just align them to the reference sequence (in your case, the entire human genome has been sequenced). Targeted resequencing means you're sequencing a specific region, such as a gene. This requires concentration of the specific DNA you wish to sequence and can be done by PCR amplification (using primers that flank your desired sequence) or hybridisation (using probes complementary to your desired sequence that are fixed to a surface), among other methods.
For information on methods for enriching a DNA sequence, give this a read:
Mamanova, L. et al. Target-enrichment strategies for next-generation sequencing. Nat. Methods. 7, 111-118 (2010).

So, let me get this straight: we PCR amplify the region of interest. Then use straight forward DNA sequencing on the PCR'd region?

Yes, basically.