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How does alternative splicing actually work?
How do two identical pre-mRNA molecules give rise to two different mRNA molecules after alternative splicing? They contain the same introns and exons and thus same splicing enhancers and silencers
Is it just a case of what proteins/snRNA happen bind to the mRNA first?

A mRNA molecule is composed of introns and exons. All introns are removed before translation occurs*. However, sometimes exons are also cut. This leads to different mRNA molecules, and therefore, different proteins. This image gives very good summaries in my opinion (source):
According the National Library of Medicine, the different mRNA strands are called transcript variants, splice variants, or isoforms. I admit that I do not understand how a cell 'decides' when to use one variant over another. This free article is on that very topic, but it is beyond me. To quote the abstract:

Regulation of alternative splicing is a complicated process in which
numerous interacting components are at work, including cis-acting
elements and trans-acting factors, and is further guided by the
functional coupling between transcription and splicing. Additional
molecular features, such as chromatin structure, RNA structure and
alternative transcription initiation or alternative transcription
termination, collaborate with these basic componentsto generate the
protein diversity due to alternative splicing.

*Some may argue that this is not true. There is a mode of alternative splicing where introns are left in called intron retention. Personally, I think that a region that is sometimes removed from the mRNA and sometimes left in should be classified as an exon, so intron retention is the results of misclassifying an exon as an intron rather than a genuine biological phenomenon.

But presumably the poster knows the part of your answer that says what alternative splicing is, and you haven’t answered his question which relates to how this is controlled, except from the quotation which you admit not to understanding. In fact the quotation says nothing of substance, but is so much hand waving.

It was not clear to me how familiar the OP was with alternative splicing, so I provided a basic overview  and a link to very detailed paper that went over the details I was not confident trying to explain. If they were new to the topic, then they have their basic answer. If they were knowledgeable in the topic already, then they have a great resource on where they can do more research. If you dislike my approach, perhaps you should leave an answer to the question.


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When does a fetus become conscious?
I thought of asking this question in the Psychology & Neuroscience stack exchange, but I ultimately decided this would be a better stack exchange for it. When do the "lights turn on" in a fetus, that is, when does it come into existence as a conscious being? I have heard stories of people remembering things while in their mother's womb, so presumably they were conscious of it, even if very faintly. So, does consciousness begin at birth, or before, and if so, how much before? I have heard that brain activity happens in the fetus some weeks after conception, so does that mean it is conscious then, however dimly?

Which of the many extant definitions of consciousness are you referring to? Even legal definitions vary with jurisdiction. Can you edit to be specific.

Sometimes I think back on how I perceived certain things as a child and think that I was perhaps barely even conscious then. One issue is that your question seems to be associating memory with consciousness. Just because I don't remember eating breakfast last week doesn't mean I wasn't conscious then. Perhaps I wasn't. Who knows? I can't remember after all.

@JiminyCricket I am defining consciousness as the capacity to experience anything, be it a sight, or a sound, or a touch, or some other sensation.

A bacterium can respond to the chemicals.  Is that not consciousness by your definition?

By that definition, the gametes are conscious before they even meet, so prior to conception presumably.

Probably need to start over at Philosophy to ask if consciousness exists. Your definition is insufficient at best. What does it mean to "experience" something? Is it merely sensing of one's environment? Does a plant sensing and responding to stimuli exhibit consciousness? Maybe. Is "brain activity" pre-requisite to experience? Is a cultured synapse firing in a petri dish conscious?

Basically, the upshot is that it seems biologists don't have the foggiest idea when anything becomes conscious.  There are still arguments about whether various animals are conscious - and I mean things like fish or insects, not microbes or other things that don't have an obvious nervous system.

It might work here if you change the parameters of the question a bit. Were you to ask what the developmental milestones are which mark a qualitative and/or quantitative increase in awareness or neural activity... You'd need to specify an end-date as of course it continues into adulthood - and at a range of different timescales per individual. Of course, you'd need to show prior research as per the [help]'s [ask] section. I'm sure you could get a fairly comprehensive and factual answer then, without all the equivocation about definitions.

source: Stack Exchange

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

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How many proteoforms are theoretically possible?
Initially, I was going to ask how many proteins were possible. But, while researching the question, I learned the word proteoform and have been reading a lot of stuff I don't really understand, but from I do think I get,  the word "proteoform" was created because sometimes the "typical" or "canonical" forms of a "protein" can take different forms, or "proteoforms" based on environmental and possibly other factors, despite being coded for with the same DNA. So, a "protein" will often (always?) have several different "proteoforms". As I understand it, it is part of the protein-folding problem, and the complexity is huge. I was trying to see if anyone has calculated or figured out what would be needed to calculate how many proteoforms were possible, but I don't find anything. I imagine the number is tremendous, I just wanted to know how tremendous. So, how many proteoforms are possible? If that's not known,  what would be needed to know this? Thanks.

Possible duplicate?: https://biology.stackexchange.com/questions/108908/is-there-are-theoretical-limit-to-the-number-of-proteins-possible-and-their-resp I know this is about proteins rather than "proteoforms", but the distinction does not seem particularly important at scales like this.

@BryanKrause I think the question is a duplicate, at least in the sense that the question+answer note the existence of isoforms/alternate structures making the upper bound of the answer huge.

Does this answer your question? Is there are theoretical limit to the number of proteins possible and their respective structure?

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


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gene dosage vs copy number
In the online articles I'm reading, I see the authors mention gene dosage and copy number. My confusion is regarding if the two terms mean exactly the same thing - number of copies a gene occur in the genome?

They don't always mean the same thing, as gene dosage may be measured by RNA-Seq, whereas copy number by DNA sequencing / other means. But people do use it as rough substitutes. Often people do try to infer copy number from RNA-Seq, as samples tend to fall in "digital" intervals of expression, and use CNV to estimate gene dosage.


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How bacteria respond to toxic viral proteins?
The lysis-lysogeny state of bacteriophage lambda is well known. Under certain conditions, the phage will enter the lysogenic state after infection of a bacterium. Then, after a while, the phage switches to the lytic state and breaks the bacterium. I forgot the details but the phage must expressed some proteins toxic to the bacteria, e.g., a protease, to lyse the bacteria.
Here is the question: Did bacteria have any immune strategies to defend themselves in face of these toxic proteins? In mammals, both nucleic acid and protein can induce immunological responses and activate T/B cells. In bacteria, I know that the CRISPR system is an immune strategy to degrade viral nucleic acids, but seemingly not proteins.
So if the CRISPR system has missed cutting the viral DNA, the phage has switched to lytic state and begun to produce toxic proteins, did the host bacteria still have weapons to defend? Obviously, most of the time the bacteria failed and died. However, after billions of years' evolution, how could bacteria just give up in face of the toxic proteins?

I don't know of any direct defenses available to bacteria once a prophage genome has been integrated. If a prophage has induced and carried out its program to the point that lysis proteins are being produced, it is far too late for the host cell.
However, an indirect defense for the bacteria is to simply survive and replicate faster than its prophages induce. You seem to be thinking about this as a purely antagonistic relationship, but once the viral genome is integrated into the bacteria, their reproductive fitness becomes intertwined. When the host replicates, both genomes are copied, so the phage can produce far more progeny by delaying activation until the host builds up its numbers. Phage that boost the fitness of their lysogenic hosts will be even more successful. Prophage can defend against infection by other viruses, confer antibiotic resistance, or provide toxins against eukaryotes or rival bacteria. After many generations, mutations that destroy the prophage's ability to induce and kill the host cell but preserve the beneficial genes are selected for; bacterial "domestication" of integrated viral genomes appears to be common. A thorough discussion of bacterial/prophage co-evolution could fill a textbook, but is mostly beyond the scope of this question. The links in this answer should provide a good starting point if you'd like to know more.

Thanks very much for your inspiring answer! Actually I wanted to seek molecular tools for proteins as CRISPR, the bacteria's immune system against nucleic acids, has been so useful.

Yes I realize my answer doesn't really address the main part of your question. I don't think there is anything like CRISPR for proteins.


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Will lidocaine stimulate photoreceptors?
Lidocaine apparently blocks voltage-gated sodium channels in sensory neurons and thus prevents action potentials, but photoreceptors like rod cells require hyperpolarization (through blockage of its sodium channels) for the neurotransmitter to be released / generator potential set up, so would adding lidocaine to the retinal cells stimulate them? Would it hypothetically make us think there's light even when it's dark?

See Wikipedia for an overview of the signal transduction that occurs in photoreceptor cells.
Briefly, light results in increases in cGMP; cGMP binds a cGMP-gated sodium channel (not a voltage-gated sodium channel) causing the channel to close. Therefore, light -> less sodium entry.
Lidocaine blocks a different type of sodium channel: the fast voltage-gated sodium channels involved in neurotransmission. It's possible there is some cross-reactivity, but it would be entirely coincidental as it's from a completely different family of channels.
In any event, the retina has all sorts of other neuron types: bipolar cells, retinal ganglion cells, and various interneuron types, that all communicate with each other through action potentials mediated by voltage-gated sodium channels. If you were to apply lidocaine to the retina, you'd lose all this neurotransmission.

the last paragraph really hit the nail, I feel like I should've realized that before.

@Rayan Good, but paragraphs 2 and 3 are probably more important to the specific question asked here: it's important to recognize that there are different types of channels that conduct the same ions, this will come up in a lot more situations.


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Herbicide resistant crop plants

Consider the above multiple choice question. I can't seem to understand why the answer is B. How does genetically modifying the crop plant have any effect on the weeds?
The way I understand genetic engineering is that we insert a desired gene into the crop plant that allows it to become herbicide resistant. So how would this gene make its way to the weed? Could someone please explain how the two effect one another?

Why would you want herbicide resistance in the crop? How would you grow the crop to make use of the herbicide resistance? What is the treatment that you're presumably applying to both the crop and the weed? What are long-term results of that treatment? Try searching the internet a little bit; we usually ask that of people posting questions here, to show some evidence of research effort.

"So how would this gene make its way to the weed?" That doesn't really matter for your question. (Evolution - selective pressure - is the answer to that concern. It is exactly like antibiotic resistance.) Just looking at the information presented, look at the advantages. What are they? What would interfere with the advantages? That's your answer.

@MaximilianPress Herbicide resistance is desired so farmers can wipe out weeds without damaging crops. So we apply the herbicide to both the weed and crop so over the time the weed may mutate to become resistant? Could this be right?

@user73245 can you find any supporting evidence for that chain of logic on the internet, wikipedia, scholarly articles, etc.?

I am pretty sure that the gene is not transferring directly from the crop into the weed. Instead, the idea is that you would never engineer an herbicide-resistant plant unless you were planning to apply herbicides to it. Since weeds will experience the effects of the herbicides along the GM plant (this is what kills them, after all), they will experience a selective pressure that may cause them to become herbicide-resistant.

What makes you sure that horizontal gene transfer doesn't occur in this case? Could you add a reference - else risk downvotes/deletion. (I'd assume the same thing as you because of the timing of the application - if before flowering and seed development, but we like references.)


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Does the X/Y sperm ratio in male ejaculates depend on circumstances of sex and who they are mating with?
Male mice are known to have different X/Y sperm ratios in their testis. Possibly, this phenomemon is not limited to the mice. However, can the males utilise that to their advantage?
For example, produce ejaculates with higher Y sperm concentration when mating with a female they have not mated before. Or when mating with a female who they perceive as more masculine (regardless of how they do that, be that pheromones, appearance or other signs).

The Trivers-Willard hypothesis says that females should change the ratio of female-to-male offspring based on her condition (https://pubmed.ncbi.nlm.nih.gov/27170721/). Maybe something similar is happening here, where males ejaculate more X-chromosome carrying sperm when in better health and less when in poorer health. I have never heard of the TWH being applied to males though, so that is honestly just my best guess.


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Why are monocots more advanced than dicots. Shouldn't more complexity mean more evolved?
Features like pith, endodermis, pericycle, medullary rays and secondary growth are not present in monocots. How are monocots still more advanced?

”more advanced” seems subjective, here. Can you link a source for that claim or provide more explanation?

After seeing hutchinsons classification this question seems wrong , the categories of monocot and dicot are not correct to group 'types of angiosperms' and to conclude that one of the groups is more evolved or primitive . Sometimes it's the plants of monocot which happen to be more developed while it's dicots in some cases .

short answer no advanced is subjective, to a beetle having an exoskeleton might seem more "advanced"

Yeah , for a land animal  it is strength which helps it in fight  situation .while for a bird which has less land animal enemies due to its flight , having strength is not useful for some birds and hence some  are small making flight easier and have bright colours to attract mates . These two traits can't be compared since they are developed depending upon the different environment they live in


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Why does plasmid recombination require precise "copying and pasting" but microinjection doesn't?
Just learning about biotechnological techniques of gene transfer at the moment. With bacterial plasmid recombination, from a high-school level, we are taught that:

The desired gene is cut using a particular restriction enzyme
The plasmid from the host is also cut using the same restriction enzyme
The desired gene is inserted into the space that was cut out from the plasmid.
The plasmid is put back into the bacteria and the gene can now be expresesd.

This makes logical sense.
With microinjection, however, we are taught that the gene is just directly injected into the cell using a needle and that it somehow manages to use it? How does that actually work? How does the extra gene just somehow get put into the existing DNA without cutting out a section like you have to do in plasmid recombination? Furthermore, if you can just insert the DNA into the cell with microinjection without all of the hassle of precise "copying and pasting" done in plasmid recombination, then what is the point of using plasmid recombination?

You can't microinject a bacterium for one thing - far too small, so you need all the components in one place to get the gene expressed and make the plasmid replicable.  This will include things like promoters, start sites, Kozak sequences, terminators, resistance genes, ori, etc.
Genes to be microinjected need, at a minimium, sites to make them insert into the host genome by recombination, or need similar components to a plasmid; promoters etc. It's not like this isn't also a precise science in how to get it to work, it's merely that microinjection is one of the techniques used to insert genes into the nucleus of eukaryotic cells.

You can't really determine the exact location of where the insertion will be, and I believe the microinjection you're talking about is using transposons (e.g. P elements).


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Why aren't iPSC's created in vivo?
As I understand it, induced pluripotent stem cells (iPSC's) can be made using the following (grossly oversimplified) steps:

Take some skin cells (fibroblasts) from the host
Introduce the transcription factors Oct3/4, Sox2, Nanog, and Lin28 using retroviruses to reprogram them into pluripotent stem cells
Reintroduce the reprogrammed cells into the host

I do not understand why steps 1 and 3 are needed, and why the retroviruses can't be introduced directly into the target area to create the iPSC's in vivo. My guesses are:

The culture conditions within an animal body are not sufficient for reprogramming or differentiation (low methylation?)
The retroviruses would die off before they could deliver the transcription factors uniformly enough or in large enough numbers for any meaningful regeneration to occur, either due to the immune system or simply because they are not self-replicating
Because the cells would not be transfected at exactly the same time, and would already be surrounded by many differentiated cells, a newly created transfected cell may immediately differentiate based on its surroundings, causing no noticeable difference compared to the cell being made through un-transfected cell division
I am misunderstanding how reprogrammed cells act, and all reprogrammed cells form a blastocyst from which the actual useful stem cells are harvested
Using the retroviruses directly could cause organs cells to become transfected and differentiate in unexpected ways, harming the host

I am not a biologist so please feel free to point out any and all misunderstandings I may have shown. The transcription factors mentioned are the ones which work on humans according to what I read, but this question is not about humans specifically.
Edit: this question appeared in the Related tab after I posted my question, but wouldn't immunosuppressants or more infectious/illusive vectors circumvent the immune system issue?

I don't know which specific barriers are the most important, but certainly it is not simply trivial to say "immunosuppressants would circumvent the immune system issue". Immune suppression is a terrible, terrible condition to induce. It's done when the alternative is death in short order. That doesn't mean that some people don't live long, productive lives on immune suppressants after e.g. organ transplants, but it's not something you'd choose for anyone that didn't absolutely need it.

But assuming it was induced temporarily (long enough for the targeted tissues to have the transcription factors delivered) in an isolated environment, would it still be effective? I'm not trying to imply it's something to try on a whim any more than if I had said "radiation would circumvent the issue of cancer cell replication", I'm just listing possibilities (not solutions) based on my limited understanding.

Pluripotent stem cells, regardless if they’re of embryonic or iPSC origin, will form a very nasty (and often very deadly) form of cancer called teratoma when present in adult tissue (Wesselschmidt 2011, Choi 2015).
I read from your question that your goal is to regenerate tissue. Although iPSCs were a breakthrough in understanding stem cell biology in general, their use in regenerative medicine is still very challenging, since their embryonic-like state is too multipotent and dangerous. Even adult stem cells (like mesenchymal stem cells, hematopoietic stem cells) are undergoing vast changes until they reach their terminal state. And even they need specific signals to develop into the right tissue type. Even when transplanting stem cell grown organs, one has to make sure that every cell is terminally differentiated, otherwise your new liver might grow something random like a set of teeth. People try to circumvent the pluripotent state to solve this problem (Pushp 2021).
Together, you were right with your last point: even if the direct retroviral transfection would work, there won‘t be directed tissue regeneration but random organ growth.


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What are the main challenges for developing imaging techniques for cellular-scale structures?
Small disclaimer: I'm not a biologist, so I'll try my best to write a high-quality, objective question, but please forgive me if it doesn't turn out as expected.
Imaging technologies such as MRI and CT Scans are used routinely to inspect anatomical structures inside the body of alive patients, and, as such, are powerful diagnostic tools. A typical MRI machine will produce volumetric images with a voxel width of 1,000,000 nm. This is more than sufficient to identify a broad class of issues, such as tumors and aneurysms. Yet, given that a human cell has about 10 nm in diameter, that resolution is 5 orders of magnitude below what would be required to image individual cells in vivo. As such, MRI, and the like, can't be used to diagnose macular degeneration or hearing loss by imaging the damaged retinal or inner ear tissues, for example, and thus rely on less precise exams, such as hearing tests. At the same time, in vitro, technologies such as electron microscopy are capable of imaging individual structures inside cells, and are, thus, capable of reaching resolutions that are impossible for live patients. If we had some hypothetical imaging technique capable of inspecting individual cells, it would enable a wide range of conclusive diagnosis for conditions that, currently, are still hard to track.
My question is: why is there such a gap, and how far we are from developing technologies capable of imaging cellular-scale structures inside the body? For example, would it be possible to create a nano-MRI machine capable of imaging someone's organ of corti, or individual retinal ganglion cells? If so, what are the relevant advancements in this direction? If not, what are the physical or practical limits that make it impossible?

I think the question would be better focused on just MRI, and possibly a better fit over at Physics. It doesn't make much sense to compare MRI, which images through tissue using magnetic fields, to something like electron microscopy that requires you to slice into something and apply super carcinogenic chemicals to increase contrast. They're not in the same realm of approaches.

https://physics.stackexchange.com/questions/621545/why-does-mri-magnetic-resonance-imaging-have-high-contrast-and-spatial-resolut https://physics.stackexchange.com/questions/111770/how-specifically-does-an-mri-machine-build-an-image-from-received-radio-waves


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Are there internal systems of organisms which acquired the functions which they were not evolved to perform?
In his research on the issue of belief formation Daniel Gilbert (1991. How Mental Systems Believe) claimed that cognition "is an evolutionary outgrowth of [perception]." What he meant is that the cognitive system processes propositional information the same (or similar) way the perceptual system processes visual images. I assume his claim implies that, apart from some of its components, the cognitive system of comprehension of communicative stimuli (utterances) uses the same subsystems of the brain which are used by the perceptual system to comprehend visual stimuli.
If my interpretation is correct, from the evolutionary point of view comprehension of the above-mentioned types of information is embodied in the same neural structures. Given that visual perception precedes perception of language, we can plausibly conclude that there is a neural structure in the brain which had evolved to process perceptual stimuli which later also begun to function as a system of processing of verbal stimuli.
Regardless of the correctness of this view, I wonder if there are any examples of the systems of an organism which were evolutionarily formed to perform some specific type of tasks and subsequently acquired an ability to perform another type of tasks? Could anyone advise on some literature related to this topic?
P.S. I hope it is clear that I don't mean the sort of acquisition of new functions which can be attributed to our motor system, which was formed to perform such functions as walking, running, grabbing, throwing, etc., and now is used to play musical instruments or paint pictures :)

Converting my comment to an answer because apparently it was what OP wanted.
The technical term for this is a spandrel, a term borrowed from architecture in a classic paper by Stephen Jay Gould.
There is considerable debate about what organismal features exactly might serve as spandrels, which is difficult to decide due to the issues with the fossil record. Examples cited in the wiki page might include the human chin, or language. However if we consider exaptations more broadly (another possible term that includes spandrels), there is potentially a very wide set of traits that might be considered spandrels.

The answer indeed helped me a lot, although the question touched a little bit different aspect of the issue. What is really useful in the approach of Stephen Gould is his critique of what he called adaptationism, which claims that every trait of an organism has some adaptive features. My hypothesis, based on the Gilbert’s account of belief formation, is that some traits are not adoptive, but are emergent characteristics of systems of organisms which really are adoptive. Recently, I have read a similar point made by Jonathan Heidt regarding reasoning and moral judgement (see the next comment).

“Most propose that the building blocks of human morality are emotional [...] and that some early forms of these building blocks were already in place before the hominid line split off from that of Pan 5 to 7 million years ago. Language and the ability to engage in conscious moral reasoning came much later, [...] so it is implausible that the neural mechanisms that control human judgment and behavior were suddenly rewired to hand control of the organism over to this new deliberative faculty.”
(Heidt, J. (2007) The New Synthesis in Moral Psychology. Science 316. P. 998)


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Cross correlation metric in ChIP-seq experiments
I found this explanation of cross-correlation metric in ChIP-seq.
Now, the definition of Pearson's correlation coefficient between two random variables $X$ and $Y$ is $$\rho_x = \frac{Cov(X,Y)}{\sigma_x\sigma_y}$$ where $Cov(X,Y) = \frac{1}{n}\sum_{i=1}^n(X_i-\mu_x)(Y_i-\mu_Y)$ and $\sigma_x = \sqrt{\frac{1}{n}\sum_{i=1}^n(X_i-\mu_x)^2}$ (similarly for $Y$).
Given this definition, I am not sure what is being computed when the above link says:

Strand cross-correlation is computed as the Pearson’s linear
correlation between the minus strand and the plus strand, after
shifting minus strand by k base pairs

Following my above notation, what would $X$ be? What would $Y$ be?

I'd read that page in a little more detail. They give an intuition here:

The concept is that if you have a read that maps uniquely on strand x at position $i$ (where $i$ is the starting position); it follows that you will have a read mapping to strand y at position $i+r$. Because the way the counts are stored, with the number of reads starting at each coordinate, you will get a bunch of reads at $x[i]$ and a bunch of reads at $y[i+r]$ that are $r$ distance away from each other.

It's not super well explained, but one presumes based on the explanations that relevant values of $r$ are the read length (how long individual reads are reading off the fragments) or the fragment length (how big the pieces of DNA that go into sequencing library preparation are on average, usually due to sonication or nucleosome positioning, based on how the cells are treated). Thinking about how ChIP-Seq works, you expect that the DNA is in a bunch of fragments of some length, and then you sequence each end of the fragment (of a chemically induced length) on opposite strands with reads (of a technologically specified length):
read1     >>>>>
fragment  --------------------- (length r)
read2                     <<<<<
positions i                   i+r

So you expect that at a certain distance corresponding to the average fragment length, there will be a correlation between reads mapping to the + and - strands some distance apart.
In terms of the statistical method, $X$ and $Y$ are the counts of reads at every position ($X$) and the counts of reads on the opposite strand at every position + some value $r$ ($Y$). Looking at their docs, you then vary $r$ systematically to obtain various datasets $X$ and $Y_r$ (a different $Y$ for each value of $r$) and see at which values you obtain maximal correlations, as shown in their figure where the X axis is $r$ and the Y axis is the corresponding correlation value:
$r$" />
Update
I am less clear on why there is the artefactual "phantom" peak of read length (in addition to the more interesting peak from fragment length), as they call it. That is not well explained by the resource that you linked. I attempted to search the site you linked for more information about the utility of the cross-correlation method, but I was not able to find anything relevant. I find it plausible that read length would introduce some sort of signal, but the mechanism is not obvious (to me).
But as far as I can tell, fragment length is the quantity of interest that is most readily apparent from this method.
If you are interested in the mechanics of paired-end sequencing library preparation that lead to the architecture shown in the monospaced text figure, I would suggest looking into documentation on that subject (one random example from Illumina). If you are interested in chromatin immunoprecipitation (ChIP), which forms the input to the sequencing library prep, I would suggest going and looking at wikipedia. Those are rather different and more fundamental questions, regarding which there exists abundant reference material.

Thank you so much - that is very helpful! That is why there is a peak at the fragment length, for example - because it is when the shifted read1 overlaps most with read2?

Moreover, how is read-length different from fragment-length? You seem to use it interchangeably but the graph has two different labels for them.

Lastly, from what you wrote, I am assuming that read1 and read2 are on opposite strands, rather than the same one? You wrote this is because of how ChIP-seq works, but I was also curious about why this is the case, in case you can help me understand that too (as I have highlighted better here - https://biology.stackexchange.com/questions/110658/questions-about-chip-seq-reads-and-macs). Thank you again - you have been very helpful!

see update @AryanDugar


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Mushroom identification, Hungary
I've found these growing on regular lawn, near some pine trees. In Europe, Hungary, Szada, near the end of October. Could you please help identify it?

Particularly for fungi, treat any ID with caution - don't eat until confirmed by a local expert.


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How does the cell-wall ultrastructure of gram negative bacteria justify it being unable to Sporulate?
The following is a quote from my Microbiology Lecture PPT: "Some gram positive bacteria but NEVER gram negative ones produce spores under harsh conditions".
It got me wondering why gram negative bacteria is "NEVER" involved in sporulation? At this level of which the argument has been presented, I deduce that the distinction should be in the cell-wall ultrastructure differences (gram positive vs gram negative). I do understand the ultra-structural differences between gram positive and gram negative bacteria but I cannot relate it to Sporulation, that is, how does the cell-wall ultrastructure of gram negative bacteria justify it being unable to Sporulate?
Before you mention it, yes I did approach my professor about it, and she, too, got interested to know about this and we are both on a quest to find an answer for such a seemingly interesting question.
Many thanks in advance!

What do you mean by "justify"?

Provides a scientifically plausible reasoning for why gram negative bacteria cannot sporulate with the reasoning being attributed to the cell wall ultra structural differences (vs. gram positive bacteria). Hope this helps. @BryanKrause

Endospore formation is probably unique to a single phylum (Firmicutes) and requires something like 80 different genes. Non-Firmicutes spore-formers (just a handful of Actinobacteria that I'm aware of) use a substantially different mechanism resulting in different spore structures lacking certain components essential to endospore formation. Seems like a bit of a stretch to assume the cell wall structure is the primary limitation.


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Questions about ChIP-seq reads and MACS
I had a few questions raised by the below diagram in relation to how ChIP-seq reads look like (source). I would be very grateful for your insight on them!
From the below diagram, it seems that, in a ChIP-seq experiment, we get information about the binding site in relation to both the Watson and Crick strands. That is, from our experiment, we get reads complimentary to both strands.
(Q1) I suppose this is because proteins bind to both strands of DNA, so that in our ChIP-seq experiment, we ultimately sequence reads from both the Watson and the Crick strands? Is that true?
Also, my impression from the MACS paper was that "ChIP-Seq tags represent only the ends of the ChIP fragments, instead of precise protein-DNA binding site" (Background).
(Q2) Therefore, shouldn't it be that we get reads mapping to either side of the binding site on BOTH strands? That is, we get reads more towards the 3' end of the Watson strand, and reads more towards the 5' end of the Watson strand, and similarly for the Crick strand.
(Q3) The diagram below shows we only get reads more towards the 5' end of the strands. Why is that?
(Q4) Finally, I know that MACs shifts reads by some bases towards the 3' end for each strand, in order to interpret the precise binding site location. It seems to assume what I am asking about in (Q3). However, if the answer to my (Q2) is "yes, we do get 4 such clusters of reads", then why doesn't MACs account for that?

Some transcription factors bind to a DNA promoter sequence near the transcription start site and help form the transcription initiation complex.  Do you mean transcription factors (TFs) in your Q1?

"ChIP-Seq tags represent only the ends of the ChIP fragments, instead of precise protein-DNA binding site" (Background).

(Q2) Therefore, shouldn't it be that we get reads mapping to either side of the binding site on BOTH strands? That is, we get reads more towards the 3' end of the Watson strand, and reads more towards the 5' end of the Watson strand, and similarly for the Crick strand.

Sequencing-by-synthesis (the approach used by Illumina which is the most common tech for ChIP-seq) always proceeds 5'->3' because of the way DNA is synthesized. So you read off only the 5' ends of the fragment, but not the 3' ends.
(More precisely: the 3' ends are the template, so the 5' is what you see in the read.)
Thus you only expect (start positions of) the reads to be at the 5' of each strand, as shown in the figure. So we only get 2 "clusters of reads", as implied by the figure and the MACS workflow, which only shifts 3' as you say.


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When I use isopentane to freeze tissue, which kind of purity should I choose? GR, AR, or CP?
GR: Guaranteed Reagent
AR: Analytical Reagent
CP: Chemical Reagent

I want to use  mouse liver, brain and freeze the complete organs at -80℃.

You would like to prevent the formation of ice crystals?

Depends on what you are doing with the frozen tissues. If you're flash freezing for histology, it probably doesn't matter. If you're doing some kind of metabolite extractions for analysis, you'll probably want to use a reagent suitable for HPLC or mass spec (probably AR in this case, but many manufacturers will sell products described as HPLC grade).

Those reagent 'grade' labels are done by manufacturer and are not standardized; see some examples:
https://chemistry.stackexchange.com/questions/575/what-do-the-different-grades-of-chemicals-mean
https://www.emdmillipore.com/US/en/lab-technical-resources/grade-definitions/c9Ob.qB.r7MAAAFEgSYWq.DI,nav
https://www.avantorsciences.com/pages/en/grade-definitions
https://www.westlab.com/blog/2018/04/06/a-guide-to-chemicals-and-chemical-grades
Some companies break these down into very fine scales. Personally, I think about them in roughly 3 grades: 1) ultrapure reagents that are the best of the best, used for the most delicate protocols; these come with a huge price premium, 2) normal academic laboratory grades that are as pure as is efficient - the specific purity depends on the chemical and how separable it is from contaminants, and 3) junk grade for budget experiments in teaching labs. I'm sure other people will bicker with me over this, but it's the system I've always kept in my head when ordering. I'm not certain in all cases that the actual contents you will receive are any different among some of the grades, depending on manufacturing practices it's more about what has been tested.
I don't know if your three grades listed are going to fit into these three categories that I personally came up with, but I'd probably put your GR and AR together in that second/middle grade; CR might belong in the third.
That said, I don't think the purity is going to matter much between those grades for your use; don't spend 3-10X the cost to get something more pure, I'd select something from my "middle" grade. Your samples themselves aren't "pure", and you're not really using isopentane as a reagent in a reaction but instead as a liquid interface for transferring temperature. Higher grades are probably more free of water and other contaminants, but the stuff you pour out is going to get filled with (small amounts of) water from the atmosphere anyways while you chill it. I've never seen a freezing protocol that was explicit about needing to use a very high purity of isopentane.


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Are there limitations in using DNase I hypersensitive sites (DHSs) to identify candidate enhancers?
Candidate enhancer regions are often defined in studies by DHSs and/or certain chromatin marks. I was wondering if DHSs are exhaustive for identifying possible enhancer regions, and if there is any work out there demonstrating enhancer regions in areas not sensitive to DNase 1.


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If we cloned an extinct animal, what would become of its gut biome?
If we cloned an extinct animal like the mammoth, what would become of its gut biome?

Seems like it would depend on the animal rather than an absolute answer that applies to everything.

Got like seven questions here. Would be easier to give a straight answer if you narrow it down a bit.

any one of these questions would be a good question, but you need to pick one.

The gut biome would be populated from whatever the environment was of the newly "cloned" animal.
Presumably the baby animal would be born by birth from a live host mother, which would be the likely source for microbiota, similar to other animals.

I think this is about as good of an answer to this question one can givd. But out of curiosity: is it really safe to assume that any microorganisms capable of populating the gut of the host species, are also able to populate that of the (extinct) cloned species? Are species separated by (say) tens of millions of years of evolution typically able to host similar microbiomes?

@gaspanic I think that we can expect there to be some dropout relative to the mother. But that's true even of mother-child pairs of the same species! For example, the fecal/gut/vaginal microbiomes of the human mother are contributors to neonatal gut microbiome. that doesn't mean that the infant gut is exactly those communities! As they say in ecology, "everything is everywhere but the environment selects", which isn't exactly true but is a good way to think about colonization.

Right, I agree that mother and child will by no means have identical microbiomes. I was more thinking in terms of whether co-evolution between host and microorganisms could have led to some of the microbiome species being directly incapable of thriving in the "cloned" species gut. But maybe it's too far-fetched to think that a "cloned" species that is similar enough (evolutionary speaking) to develop in its host, would be different enough to significantly affect the ability of some of the host's microorganisms to colonize its gut.

@gaspanic there is some work on coevolution of gut microbes and host, but I find the work somewhat unconvincing as a general rule. There are some specific hybrid lethalities in wasps driven by microbiomes, and a little evidence for human genetic variation associations, but it seems very case specific. Mice will grow transplanted human microbiomes just fine.

Thanks @MaximilianPress, will check these out! Not my field of expertise.


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What kind of filter can be used to filter sterilize ADC?
I am using this recipe to mix my own ADC (Albumine+Dextrose+Catalase), without the catalase for a microbiology application.
It says use a filtering apparatus to sterilize the ADC because it can not be autoclaved. We tried using a vacuum filtering apparatus with both 0.22 um and 0.45 um PEF filters, but they clogged immediately.
We have successfully used the same filters to filter other reagents in our lab.
What kind of filtering apparatus/material would be able to filter sterilize this recipe for ADC?


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Does the human body need fat for other uses than to cover essential acid requirements?
Linoleic acid and alpha linoleic acid are the two essential fatty acids, from which other fatty acids can be synthesized internally. See for example here: Essential fatty acids. Given that the requirements of these fatty acids are met, what other function do dietary fats provide which are unique to this macronutrient?
To clarify I'm asking about functions that must be provided by dietary fatty acids that cannot be provided by fatty acids synthesized from acetyl CoA from other dietary sources.

Surely by the definition of the word, essential, there can be none regarded as unique.

I have heard about fat being necessary (?) for the absorption of certain vitamines

But this is none-specific, and fatty acids can be synthesised from acetylCoA generated by the oxidation of carbohydrates etc.

You really need to clarify your question if you expect anyone to answer it with or without a bounty. Are you asking whether there are functions that must be provided by dietary fatty acids that cannot be provided by fatty acids synthesized from acetyl CoA from other dietary sources? (Incidentally, do you know what acetyl CoA is?) we can help you if we know what you are asking. If you don't know, you shouldn't be asking.

Fats can serve non-metabolic functions as well, for example structure and support around organs, padding or insulation.


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Is ribosome denser and smaller than nucleus. Also sone relations in centrifugation related to size and density
I want to know that if ribosomes are denser than nucleus. If so, then they should settle at the bottom of tube in centrifugation (following a density gradient) before the nucleus.
Secondly, if ribosomes are smaller than nuclei than they should settle after the nucleus (which they do) but they should also have a smaller Svedberg value (I don't know whether they do or not).
More simply, I want to know whether density or size is responsible for their sedimentation after the nucleus.
I also want to know the relationship between size and Svedberg value of organelles in general.

I think that the higher speeds necessary to collect ribosomes by ultracentrifugation are likely to disrupt the nucleus. See for example this extract from a well known textbook:

This treatment separates cell components by size and density: in general, the largest units experience the largest centrifugal force and move the most rapidly. At relatively low speed, large components such as nuclei sediment to form a pellet at the bottom of the centrifuge tube; at slightly higher speed, a pellet of mitochondria is deposited; and at even higher speeds and with longer periods of centrifugation, first the small closed vesicles and then the ribosomes can be collected (Figure 8-8). All of these fractions are impure, but many of the contaminants can be removed by resuspending the pellet and repeating the centrifugation procedure several times.

See the image they reference, which shows that the supernatant from spinning out nuclei will then yield ribosomes upon subsequent spins:

Ribosomes are much smaller! Recall that Svedberg units are indirect measures of size. I am not sure that many of the large organelles have Svedberg values per se. Even the ribosome falls apart into subunits upon ultracentrifugation after all, it doesn't really have a singular S value I don't think?


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Could abiogenesis still be ocurring?
From my understanding, most biologists believe that under certain conditions life can form from inert chemicals. If the conditions for this to happen still apply, is it possible that in bodies of water single-cell organisms could still be forming?

From the latest I've heard or read, there is too much oxygen in the atmosphere and oceans... Maybe highly anoxic places might suffice... Semi-arid areas on early land with small pools of water and underwater vents are where most scientists believe life arose... Way too much toxic/reactive O2 there now....

Welcome to SE Biology. Please search the site before posting. Searching for "abiogenesis" shows several related questions, e.g. https://biology.stackexchange.com/questions/59892/is-abiogenesis-possible-today


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What percentage of our DNA is never transcribable? What percent is never actually transcribed into RNA unless researchers force it to in a lab?
From what I gather, the vast majority (but not all) of the DNA in our genomes will transcribe and create an RNA, if only under certain conditions in a lab (forcibly unwound, among other steps).
How much of our DNA can be transcribed but not translated?
How much actually does so on a regular basis in our cells, becoming useful RNA, but doesn't code a single amino acid?  (Non-coding functional RNA, or junk RNA?)

While, as you say, most of our DNA can be transcribed, you are right that it is not well accessible and/or lacks strong promoters. It's said that over 80% of DNA is transcribed but only <2% transcribes into mRNA, that will be translated [1]. It is not yet known what percentage of non-coding RNA (ncRNA) is junk (by identity) [2], but if weighted by mass or numbers, most of ncRNA is functional as ribosomes and tRNAs make up most of the cells dry mass:

(image source)

Minor detail: doesn‘t the left pie chart say that rRNAs make up most of a cell‘s dry mass and not tRNAs..?

I once read in a book „Numbers in Biology“, that ribosomes can make up to 80% of cells dry mass. The chart is not about dry mass but about RNA mass, ignoring not only water but also lipids, proteins etc.

Oh I meant together they make up most of the cells mass. Not tRNA alone.


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Rationale for two-components in feedback loop of circadian molecular clock
The circadian clock in humans and fruit flies involves a feedback loop in which the active components are protein heterodimers. This is illustrated for humans in the diagram below:

The nuclear proteins MBAL1 and CLOCK form a dimer which is a specific transcription factor for the genes of proteins PER and CRYS. The mRNAs for the later are transported to the cytoplasm for translation, where a PER/CRYS heterodimer is formed. This is transported to the nucleus (generally in a chemically modified form) where it inhibits the MBAL1/CLOCK dimer, and hence the transcription of the PER and CRYS genes.
The diagram above is take from Ko and Takashi (2006) and a summary of the process can be found in the Wikipedia entry for Circadian Clock.
I wish to know if there is any known reason or rationale for this feedback loop involving proteins that are made of two separate components, and whether simpler systems involving a single-component transcription feedback loop exist, for example in more primitive organisms.

Heterodimer of what? You need to provide more information about the process (preferably supported by a reference source) for your question to be intelligible.

In mice models, I speak of the heterodimerization of BMAL1-CLOCK and in fruit flies this is CLOCK-CYC. These are mentioned in the wikipedia article: https://en.wikipedia.org/wiki/Circadian_clock (scroll down to Mammalian Clocks and Insect Clocks section)

One question that is implicit here is whether complex biological feedback loops exist that do not have heterodimers. Heterodimerization is a fundamental design component of biological systems in cells. I don't have a reference but I'd be very surprised to find any complex cellular system that didn't involve the heterodimerization of protein components as a step!

As an example, the arabidopsis circadian clock involves a variety of distinct protein complexes (which at times are heterodimers), some of them homologous to animal models.

I think your question is valid, but the lack of response suggests that the problem could be presented more clearly. I have taken the liberty to do so. You can roll it back if you like or edit it further.


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Nitrate fertilizer problem

Take the above problem. I understand how to do the calculation. However I can't seem to understand the units of the answer in the calculation.

The answers illustrate the units as t/ha^-2 which is equivalent to t(ha)^2. But where do these units arise from? Crop yield is represented by tones per ha in the table above. So wouldn't total crop yield just be tones? I can't seem to find any intuition whatsoever as to why the units are t/ha^-2? Could someone please give me a hint or a solution?

Please do not post text as images. Accurately type the text into your question. Images are not searchable, and cannot be interpreted by screen readers for those with visual impairments. Use the [edit] link to modify your question.

I think this is a mistake in the provided solutions – it should say either $\mathrm{t/ha}$ or $\mathrm{t \, ha}^{-1}$. Furthermore, the answer should be multiplied with the area of fields.
There are two different quantities in this task: yield which is the mass of harvested crops (and has units $\mathrm{t}$), and field yield which is yield per unit of field area (which has units $\mathrm{t/ha}$). In the provided table, you are given field yield, but in the question, the farmer wants to have the highest yield.
The yield if fertilizer is put on one field will be:
$$m=7\;{\rm \frac{t}{ha}}\times 1\;{\rm ha}+3\;{\rm \frac{t}{ha}}\times 1\;{\rm ha}=10\;{\rm t}.$$
The yield if the fertilizer is spread equally on both fields will be:
$$m=4.8\;{\rm \frac{t}{ha}}\times 1\;{\rm ha}+4.8\;{\rm \frac{t}{ha}}\times 1\;{\rm ha}=9.6\;{\rm t}.$$
Therefore, the farmer will produce more crops if he puts all fertilizer on one field.


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Why Sexual Reproduction instead of Gene Transfer?
So, as I understand it, the prominent theories for the evolution of sex are that it increased variation allowing for faster adaptation in evolutionary arms races, and that it gets rid of deleterious mutations by introducing unmutated alleles in descendants. But why sex specifically for this task? Surely the same benefits could be conferred through horizontal gene transfer. We could even conceive of a kind of non-sexual diploidy.

Why not questions about evolution are usually impossible to answer. Evolution is random and requires precursors from which to evolve. If no sexual beings had the ability to transfer genes any other way, it could not evolve. See Why do some bad traits evolve, and good ones don't?

@anongoodnurse: if you assume that there are no precursors, please provide references for that claim. Horizontal gene transfer  in eucaryotes is being discussed, while the molecular machinery for tunneling nanotubes is present in eucaryotes. Apart from that, this question is very much answerable even without knowing about a precursor, since comparing strategies for increasing genetic variation is absolutely valid and should be encouraged on this site. Referring to that thread is a lazy way of avoiding actual answers.

Bacterial conjugation does provide genetic variation, but wikipedia mentions a vast array of different mechanisms how sexual reproduction recombines genes, which seems to beat horizontal gene transfer in terms of fitness gain. Also: keep in mind that eucaryotes have a different genome architecture (enhancers, histones, genes are bigger and harder to transport), and that DNA outside the nucleus is often interpreted by the cell as a viral infection, which could make horizontal gene transfer difficult without risking immunity.

@markur - Commenters are not obligated to provide references. OPs are required to show research, and answers are required to cite sources. You are welcome to provide an answer about asexual eucaryotic reproduction, or the evolution of sexual reproduction, or whatever your heart desires (with sources.) (Also, I don't get my information from Wikipedia when it comes to science. May seem elitist, but there are just too many errors for my taste.)

@anongoodnurse - yes, the lack of sources is the main problem here (not evolution). I like to cite wikipedia in comments to encourage OPs to start their own research with simple tools. One shouldn’t be afraid of Wikipedia, it is definitely better than providing nothing and would improve the quality of many questions. Wikipedia is neither good or bad, it is what it is. Elitism won‘t attract professionals to this page but rather repel them.

@markur - Wikipedia is a fine starting point; I just don't rely on it for science. As to what attracts professionals to this 'page', why not make a proposal in meta with your insights? I'm sure more professionals (at least those with decent analytical, communication and self-appraisal skills) would be a boon to this site. You can also answer the question yourself, instead of (or in addition to) accusing others of laziness.

Let us continue this discussion in chat.


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Maximum number of chromosomes a human can have without dying?
I know that trisomia of chromosome 21 gives a person the Down's syndrome. Sometimes one can have sex-chromosome trisomia, and it is not apparent until adolescence. Certainly none of those problems kills its victim.
In theory, somebody could have both these trisomias at once, meaning they would have 48 chromosomes - 21 pairs and two triplets.
But some trisomias can be deadly (e.g. in chromosome 13 - Patau syndrome, often results in stillbirth).
So how many extra chromosomes could a human theoretically have, without dying?  I mean the total number of them.

It's not clear what you're asking. Clearly you understand that even 1 extra chromosome can be fatal, though for some it is survivable. Are you asking if there are survivable quadsomies? If there are survivable trisomies besides 21 or X/Y combinations? What's the value of this question besides trivia?

What does "the total number of them" mean?

Well there exists a condition called tetraploidy which you may have figured out means 4 sets of chromosomes in a cell. XXXX in females and XXYY in males and that's 92 chromosomes life expectancy is about an year

You need to verify your answer with a source (a reliable one). The vast majority of humans with true tetraploidy die in utero or shortly after birth. Survivors are usually mosaics. There are exceptions, but they are rare indeed.


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Electronic properties of neurons
This is about the electronic properties of neurons.
When a signal is sent from 1 part of the brain due to a stimulus,are neurons considered voltage or current sources?

Can you please provide context of what you already know and what you've read trying to answer your question? As written, it doesn't make much sense in the context of biology.

It is necessary to study the known substitution circuits of neurons - these are representations of neurons in the form of equivalent electrical circuits, where capacitances, inductances, or others imitate the potential-current properties of membranes and other conductive elements


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What microbial contaminant grows blue on L-agar/L-broth and is resistant to carbenicillin?
We are trying to grow liquid cultures of Mycobacterium smegmatis as a host for bacteriophages, but we have been having issues with contamination. Our latest attempt went like this:

Tuesday 1 pm: Added 20 ml 7H9 broth, 2 ml Albumine-Dextrose solution, 0.02g CaCl2, one colony from master M. smegmatis culture was added to 100 ml Erlenmeyer flask. Incubated in a shaker at 35° C.
Wednesday 6pm: Added 20 ul of carbenicillin (should have been added from the top, but the carbenicillin arrived a day late)
Thursday 1pm: liquid culture is turbid white and clumpy as expected.
Friday 2pm: Culture is still turbid white and clumpy. Inoculated a streak on left half of L-agar plate.
Saturday 3pm: Streak plate shows streaks of yellow-white colonies (as expected), but also a blue diffuse contaminant which smells very bad. This is similar to what we've seen in our previous attempt at growing this culture

Now, the question is: What could this blue contaminant be? The original culture protocol used Cycloheximide to control for fungal growth, but ours has not arrived yet.
A related question is. Where could it have come from?
Master M. smegmatis culture

Liquid M. smegmatis culture

Contaminated streak plate

Pseudomonas aeruginosa is a likely candidate. This is a common skin organism and cultures/colonies often have a blue/green sheen. Pure cultures generally have a fruity/ester smell.
The blue/green pigment is a combination of pyoverdine and pyocyanin.
Of note is that it is also commonly antibiotic resistant having intrinsic antibiotic resistance to a range of antibiotics, including the penicillin group


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Mathematical aspects of the study of dinosaur locomotion
I'm interested in getting a better understanding of dinosaur locomotion. Therefore, I am looking for a book that will cover various mathematical aspects and discuss various mathematical tools for studying dinosaur locomotion. I do not have a basic biological education with a specialization in paleontology. However, I am proficient in some computer algebra packages and I enjoy learning applied mathematics.
Can anyone suggest good names that give a good basic and systematic idea of the problem described above (with examples of the use of computer algebra packages if possible)?
I have tried searching for suitable sources. I came across mostly articles on a narrow specialized issue. And I would like the first sources to give me a general idea of the general methodology, so to speak.
The object of study is clear: the musculoskeletal system of dinosaurs. Subject of study: mass-dimensional properties of dinosaurs, kinematics and dynamics of their movement. I have not fully formed in my head the methods that are used for research. It is clear that these are elements of mechanics and statistical analysis. Well, that's all I know today.
Specialized articles I found:

William Irvin Sellers, Lee Margetts, Rodolfo Aníbal Coria, Phillip Lars Manning. March of the Titans: The Locomotor Capabilities of Sauropod Dinosaurs

Bohun, Sean. Mathematical model of the mechanics and dynamics of the tails in dinosaurs.

Update March 23, 2023:
After a necessary pause, I returned to the study of this topic. An image formed in my head of how the movement of dinosaurs is studied. But some things need clarification. For example, in article 1 that I cited here ("March of the Titans") there is a figure 4. I am attaching a screenshot here.

In the figure, everything is more or less clear to me, except for these red cylinders. The article (in the section Muscle and Joint Locations) describes at length what it is. But neither specific methods nor principles are given.
So my new question is about this. If respected experts will help me figure it out, I will be very happy and grateful.

What research have you done on this already? Have you looked for literature reviews or even primary literature, such as on Google Scholar? That may be a much better (and certainly more up-to-date) resource than a book. For example, look through the results of this search.

@MattDMo see my edit, please

can you edit your question to include some of the specialized articles you found? I'm guessing that you won't find books on dinosaur locomotion/kinematics specifically. However, if you search for/find articles on models for animal locomotion/kinematics more generally, that will probably give you most of the background you need to understand more specialized studies.

@BenBolker see my edit, please. Yes, this is an alternative approach - if I can't find a source where I can get the maximum information in a short time, then I will put together a puzzle from pieces of information scattered across highly specialized publications. I also came across a book by Robert McNeill Alexander. Dynamics of Dinosaurs and other Extinct Giants, Columbia University Press, 1989. I leafed through it, it's a good book, but it's pretty "old" today. And it's not as mathematical as I would like. Therefore, I did not include it in the list of references.

The cylinders will be approximations of muscle groups and/or tendon/ligaments.

@bob1 and on what principles is their calculation based, number, location, etc.?

@dtn Position will be based on anatomical clues - you can see on bones where soft tissue attaches and the size correlates with strength/stressors put on the muscle or bone attached. I don't know much more than that though as I have very little anatomy training.

@bob1 But your comment is really very important, clarifying and confirming. In one of the articles, I came across something like this: it depicted a dinosaur bone, on which possible attachment points for muscles were marked. And that was the starting point. Now I will try to find a specialist who is well acquainted with the mathematical side of the issue. I have a small request for you: the knowledge that you consider significant for the question I asked (at least the biological basis) - could you arrange it as an answer?

At OP's request:
Disclaimer: I don't have any full answers to this question or any training in anatomy beyond the basics from undergrad many years ago, so if any of this is outright wrong, feel free to correct me and either comment so that I can fix it or fix it yourself.
In the update in March 2023; OP asked what is the purpose of the cylinders and how are they determined?
The rods are approximations of muscle groups and their attachment points on the bones. If you look closely at the diagram you will see small bumps or lumps in the bone structure. These lumps are called by various names in anatomy, depending on who named them, how big they are and a range of other factors. These names include things like Processes, Tubercules, and Eminences. These bits indicate the attachment points of tendons (I remember it by: tendons are tenderer and join muscle to bone.) and ligaments (bone to bone) to the bones. There are also other anatomical markings that indicate the attachment points, but I don't know the names of them.
One might presume that, based on what we know of the anatomy of similar animals in the current day, we could use that knowledge to predict what the muscles and joints of dinosaurs look like, and how they behave. Of course, many dinosaurs were much larger than modern animals, but this is not always the case; the smallest dinosaurs that we know of are about the size of a hummingbird1, so this might not be an insuperable problem. In addition, the bones of many dinosaurs are so similar in structure to modern animal bones, that, even with a basic knowledge of anatomy, you can easily identify the major bones such as femurs and vertebrae, no matter how large (or small) they are. This means we can make some extrapolations from current animals to dinosaurs as to how the joints and muscles worked and where they were placed.
As to how the models of the muscles work, I have no idea. I do know that muscle strength doesn't scale with size necessarily, but does with cross-section2, but beyond that I have no knowledge of what this might mean for an anatomical model and how to apply it.
Refs:
1: Xing, L., O’Connor, J.K., Schmitz, L. et al. Hummingbird-sized dinosaur from the Cretaceous period of Myanmar. Nature 579, 245–249 (2020). https://doi.org/10.1038/s41586-020-2068-4
2: Jones EJ, Bishop PA, Woods AK, Green JM. Cross-sectional area and muscular strength: a brief review. Sports Med. 2008;38(12):987-94. doi: 10.2165/00007256-200838120-00003. PMID: 19026016.


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What is this small creature on the bottom of this leaf?

This first one appears to have been flattened by the slide cover.

This one is not flattened.
Probably like .2/.3 mm in size. All of them seem dead because they are doing nothing. All on the underside of the leaf.
I found these on a leaf. I am in a forested area of South Carolina. Here is an image of the tree where these are.

This is a sessile, sap-sucking insect, most likely a whitefly larva close to maturation. Like you say, they attach to the underside of leaves and don't move. Unlike some scale insects, to which whiteflies are related, these will eventually hatch into a more normal-looking fly. Here's an image of a whitefly larva for comparison:

The second image may be the abandoned skin after molting, these are usually more visible than live larva.
More photos showing more of the life-cycle and different species are available on this University of Florida website.

I had scale insect in my mind, but lack of evidence to suggest so.


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Is it true that every organ was inherited from common ancestor?
For me it seems nearly impossible that two different branches of evolution came up to same organ or feature.
Is there any proof or refutation that every organ or feature was inherited from common ancestor?
Is there any mechanism of adopting organs or features from different branches?
Example of organs and features:

Eyes
Body symmetry
Head
Lungs

This question seems relative a bit: What is the evidence that plants and animals had a common ancestor?

Eyes are a great example of convergent evolution: https://en.wikipedia.org/wiki/Convergent_evolution Even if you don't believe it, this is what happened.

The catch-all terms in your list belies the differences that exist.  There are radically different kinds of eyes. For example, your eye, insect eyes, octopus eyes, and  lobster eyes are all wildly different. The same is true with lungs (invertebrate lungs vs vertebrate lungs). This is also true with hearts, limbs, ears, mouthparts, and taste/smell organs

"Is there any mechanism of adopting organs or features from different branches?" We suspect this occurred on a microrganism level where organisms can absorb each other and organelles, but not a between larger multi-celullular organisms.  However, that kind of stuff tends to go so far back that they might not have been considered different branches at the time.

For me it seems nearly impossible to imagine the changes possible over millions of years. A dollar is a very small thing, but think how different your life would be with 50o million of them. Now think how much greater a year is than a dollar. It doesn't matter what seems impossible to you or to me. It is what it is.

what prior research have you done, eyes and symmetry have evolved independently several times. eyes are particularly famous for it since it has happened almost a dozen times. also there are no common organs among animals and plants are you perhaps confusing organelles and organs?

@DKNguyen but could all these different eyes be evolutionized version of the initial primitive eye?

@k06a See answer.

It seems that some features/organs could appear during evolution independently for different branches of evolution.
https://www.scienceworld.ca/stories/evolution-flight/

Flight appears to have evolved separately four times in history: in insects, bats, birds and pterosaurs. These four groups of flying animals didn’t evolve from a single, flying ancestor. Instead, they all evolved the ability to fly from separate ancestors that couldn’t fly. This makes flight a case of convergent evolution.

For chordates and, more specifically, vertebrates, most organs do share a common ancestry. Some organs with different functions share the same origin too. For example: cephalochordates have a iodine mucus-producing gland called endostyle, which roots back to the first protochordates. Its function is to capture microorganism and detritus.  In vertebrates the endostyle became the thyroid gland, which obviously has a different function but still secretes iodine.
However, if we take a look at Metazoa in general (=basically all animals grouped into one taxon) some organs definitely have different origins because of convergent evolution. For example, vertebrates and cephalopods both share camera-type eye and a similar central nervous system but those features evolved separately. Same thing applies for arthropod hearts and cardiovascular system compared to vertebrates’ one. Both have a closed circulatory system but that’s a certain case of convergent adaptation, since deuterostomes’ and protostomes’ last common ancestor was an acoelomate and didn’t even have a cardiovascular system in the first place.

I'm impressed there is evidence for so fundamental an necessary an internal organ as the heart, What is the evidence for complete independence for vertebrate and cephalopod and vertebrate eyes? Is there any? The eyes obviously developed along different lines but from context of OP's question, it seems even originating from a common light sensitive organelle in a micro-organism could disqualify it as being of truly independent origin.

"For me it seems nearly impossible that two different branches of evolution came up to same organ or feature."
Depending on what level of organization you are talking about ("feature" is rather imprecise), the answer differs somewhat, but in general it is quite possible and instances are known, as laid out a bit in other answers.
If you are talking about an "organ", depending on what you mean, the panda's thumb might be an example, or eyes.
What's instructive about the example of e.g. cephalopod vs. vertebrate vs. insect eyes is that while they are quite different structures, they are all actually quite similar at the molecular level, for example using homologous opsin proteins, chemical processes, and in fact arising from the same ancestral tissue lineage. The case is instructive in that the evolution was parallel, arising from a shared set of input components among all lineages, rather than convergent, arising in a totally de novo fashion (though in general people seem to prefer "convergence" as the more general term). This kind of parallel evolution is much more frequent than completely independent convergent evolution.
More generally, convergent evolution occurs more frequently between closely related lineages than between distantly related lineages, arguing that the shared evolutionary context makes it more likely that the same adaptations can occur. Similarly, such convergence tends to reuse the same molecular components rather than to derive similar components independently.

It is true as a general rule, with exceptions.

The catch-all terms in your list belies the differences that exist. There are radically different kinds of eyes. For example, your eye, insect eyes, octopus eyes, and lobster eyes are all wildly different. The same is true with lungs (invertebrate lungs vs vertebrate lungs). This is also true with hearts, limbs, ears, mouthparts, and taste/smell organs
For the eye, specifically, it would not be surprising if we were unsure whether it can be evolutionized to a single initial version of a primitive eye. The issue is that if you go far back enough we just aren't sure due to lack of fossil evidence. The simpler things are, the less variation there can be and the more likely two independent things will end up the same. If there were a common origin for all eyes, it would likely be a light sensitive organelle on the microorganism level and you can see why fossil evidence for such a thing is tricky to discern even if found it.
Even going as far back as the mysterious Pre-Cambrian may be insufficient.  Jellyfish, molluscs, and worms (our ancestors) already existed, and likely arthropods (marks that look like leg tracks have been found), all of which are known to be capable of light sensitivity...in modern times at least.
But let's not forget plants can detect light too and they go back even farther so that could be your evidence for convergent evolution of eyes...but again, it's also  not a sure thing unless you can find and compare genetic evidence for light sensitivity between living plants and animals).
But eyes are a difficult and unnecessary example if you just want completely independent convergent evolution examples. Legs are dead easy. Arthropods and "fish" diverged before the "fish" even had fins (from which limbs would evolve for all tetrapods), and arthropods already had legs.
Another easy one is wings between insects, bats, pterosaurs, and birds. Less "optional" structures such as internal organs (and especially body symmetry) tend to go back really far and make things difficult.


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Please help me identify this tree/fruit. Is it edible?
When free, could the experts here please help me identify this tree.
Location: Delhi, India
Height: 20-30 feet
Date: October
Is the hanging green colored fruit edible?
Thank you for all the kindness in Biology SE.

This looks like Catalpa bignonioides, aka the Indian bean tree and is commonly used as a garden or street tree.¹ The plant is  native to the southeastern United States but was introduced in northern India later.²
Regarding edibility:
No,  I would not recommend eating any part of it as multiple websites list it to have no edible uses, and the roots are highly poisonous³ ⁴

References:

https://en.wikipedia.org/wiki/Catalpa_bignonioides
https://powo.science.kew.org/taxon/urn:lsid:ipni.org:names:30194969-2
https://pfaf.org/user/Plant.aspx?LatinName=Catalpa+bignonioides
https://homeguides.sfgate.com/catalpa-edible-90090.html


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Is there any reagent that can help me obtain the protein in a 14 μm tissue without damaging the morphology of the tissue
I need to extract proteins, but I want to protect the morphology of the tissue. Can I use lysis buffer directly？

The morphology of tissue is due in large part to proteins. Trying to extract them with lysis buffer will destroy the tissue.


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What is the ratio of pre-mRNA to mRNA?
What is the ratio of spliced to unspliced introns in a cell? I found, that the median half-life of a human mRNA is roughly 10h [1], while transcription and splicing each take around half a minute  [2, 3]. Based on this 1200x time-ratio, my default assumption is that the ratio between pre-mRNA and mRNA of the same genes follows a similar ratio.
Does this estimate seem reasonable? Are there any more direct experiments, that address this?
The context is crosslinking immunoprecipitation (CLIP), where some fragmented (and poly-(T) purified) RNA-reads might be only uniquely mapped if limiting mapping to the the pool of spliced mRNAs, while the same read may show up in introns. Knowing the ratio between introns and exons can help to evaluate the associated risk of such a simplification and to find suitable cut-offs.

Does this answer your question? What percentage of introns are spliced in polyadenylated mRNAs?

I see that you previously asked a very similar question. I think it is a duplicate, if not then it would be helpful to know in what ways it differs, or to have a single edited question.

I agree that it could make sense to ask these questions next to each other, but IMO those are dichotomous questions, requiring entirely different knowledge to be answered: One question asks about the ratio between pre-mRNAs and mRNAs, which is a question of life-times, while the other question asks about the ratio of spliced to non-spliced introns within polyadenylated mRNAs, ignoring the fraction of non-polydaenylated mRNAs.


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What is my original skin tone i.e. my genetic skin tone with minimal/ subtle sun tan?
When I was younger (about 2-3 year old) I had a lighter skin color and now I have a darker skin color ( currently 19 years old ). Now as a teen I have became conscious about my skin issues ( including sun tan). I have recently started using Sunscreen in the hope to get closer to my original skin tone but I don't want to keep any false expectation so I want to get an idea of What my original skin tone could be like i.e. my skin tone with minimal/subtle sun tan ?
I searched through internet to get the answer and found a convincing answer on Quora but I don't trust the source also the answer mentions some facts that I've never heard of so I'm posting this question here.
Quora Answer
Here's the summary of the answer for a quick perusal:-

Most children born of darker skinned parents are lighter in color. Both because the melanocytes take some time (mostly) to really start working. Also after a while they stop protecting babies from the sun with a cloth, and let the sun change things.
So I would say your ‘real’ color is what it was at six months of age. Of course, your sun tanned skin is not ‘unreal’ either, but it is different. I believe the six months of age answer is what you are looking for.

Welcome to Biology.SE! While we encourage new users to be active and ask questions, we also recommend that duplicates are not posted. This question seems awfully similar to this one, and if the existing answer for that answers your question, kindly close or delete this question. Ensuring that duplicates don't exist guarantees a more organized website that users can navigate easily. If the link I shared answered your question, please don't hesitate to upvote the answer (and/or the question)!

@Zo-Bro-23 I read that question earlier before posting this one but these are different questions the one mentioned by you ask "Why does this change of skin tone happen in the first place? Also, is it possible to get back to that same skin tone as i was, when i was a baby?" whereas I'm confused about " What my skin tone is in first place ?" How do you find it the same ??

I get it now; you're right that these questions are different. My bad, sorry!


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Is it safe to autoclave waste at the same time as lab equipment?
We have recently moved into a lab space where we share an autoclave with other people.
We generally autoclave empty beakers, flasks, etc., before running experiments, and we autoclave waste (Petri dishes, pipette tips) in autoclave bags after our investigations.
Reading up on the proper use of an autoclave, I came across this quote:

Do not mix autoclave bags and other items to be autoclaved in the same
autoclave cycle. Liquid media requires a shorter cycle, often 15- 20
minutes, while autoclavable medical waste requires a minimum of 30
minutes in order to be effectively sterilized

But my understanding was that whatever comes out of the autoclave would be sterile as long as you pick the proper cycle.
To be clear, is it ok to autoclave waste and equipment in the same cycle? For example, liquid waste and liquid 7H9 on a liquid cycle or solid waste and empty beakers on a gravity cycle.

You generally don't want to cook your media any long than necessary to sterilize it,  depending on how sensitive your application is. Excessive autoclave times can degrade media's ability to support certain organisms. It might be less of a problem with lab-adapted strains than wild-type isolates, but still not in line with what I would consider best practices.

Short answer: no.
The reason is that waste volumes are typically variable (e.g. one beaker with 50 ml, but another with 2 litres) and often have high biological loads compared to media or similar materials. If you were to put both into the same autoclave, and sterilized the waster properly, your media would be over-autoclaved (results in sugars being caramelized, damage to other nutrients too).
As your quote suggests, wastes take longer to decontaminate because you need to be certain that the waste is decontaminated before it can be discarded into normal waste streams (landfill or incineration etc.). If the waste is not decontaminated properly, you run the risk of accidental release of unwanted or restricted organisms into the environment. You may not care if your M. smegmatis contains a genetic modification and gets out of containment, but I bet your local biological safety officer/committee do, as do the local laws surrounding this topic.
Long story short - use the appropriate cycle for differing loads.

Thank you bob. How about solid waste  such as Petri dishes in autoclave bags, plus empty beakers/flasks etc? Our autoclave is large enough that we could put the flasks and beakers on a separate shelf from the waste bags.

@JohanWikström this may be OK, but generally you want your glassware to remain clean - sterile isn't the same as clean! In addition "dry" goods cycles usually have a drying process built in to remove most of the water from the items being autoclaved. If you run dry goods through a waste cycle you are likely to end up with a lot of water in your glassware.


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Plasmids for high school students?
For a final project in AP Biology, I am planning to design an experiment that uses the plasmid #170380 from Addgene. However, under availability they say "Academic Institutions and Nonprofits only".
Is there somewhere else that will ship plasmids to a high school student?

Schools don’t count as academic institutions?

No, because I am doing this for a final, it's not a district wide thing.

Have you tried putting in your school as an academic institution? If this doesn't work, try to contact their help. It shouldn't be too problematic for you to get this plasmid. As a last resort you can try looking for papers who used this plasmid in their work and write a polite email to the corresponding author of the paper if he/she can supply you with the plasmid. Usually works pretty fine.

I’m voting to close this question because it is about the definition of academic institutions rather than biology.

I'd suggest looking at the Terms and Conditions for nonprofit use, here is a screenshot of where it is on the webpage:

Look at this form that they link to for legal understanding of what they mean:

NONPROFIT ORGANIZATION(S): A university or other institution of higher education or an organization of the type described in section 501( c)(3) of the Internal Revenue Code of 1954 (26 U.S.C. 501( c)) and exempt from taxation under section 501(a) of the Internal Revenue Code (26 U.S.C. 501(a)) or any nonprofit scientific or educational organization qualified under a state nonprofit organization statute. As used herein, the term also includes government agencies.

and

The RECIPIENT and the RECIPIENT SCIENTIST agree that the MATERIAL:
a) is to be used solely for teaching and academic research purposes;
b) will not be used in human subjects, in clinical trials, or for diagnostic purposes involving human subjects without the written consent of the PROVIDER;
c) is to be used only at the RECIPIENT organization and only in the RECIPIENT SCIENTIST's laboratory under the direction of the RECIPIENT SCIENTIST or others working under his/her direct supervision; and
d) will not be transferred to anyone else within the RECIPIENT organization without the prior written consent of the PROVIDER.

All of this suggests to me that a high school would be fine, only wrinkle I can think of is whether they think that you can decontaminate any resulting materials (i.e. put it in a biohazard bag or bleach the hell out of it).


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How did Mendel know if a plant was a homozygous tall (TT), or a heterozygous tall (Tt)?
I had some issues while studying Mendel’s rules of inheritance.
How did Mendel, while running his test crosses, know if the plants in his F0 generation were TT (homozygous tall) or Tt (heterozygous tall)?
There seems no way to differentiate between them, and I do not know of any sophisticated tools which may do so, but surely they were not available to him?
There may be some flaws in my logic, as I am a beginner, but I am not able to find any sources which tackle this problem.
I am familiar with the basics of inheritance.

Pea plants naturally self-fertilize. So a pea plant that hasn't been manipulated will be "true breeding" that is, homozygous for the allele in question. You can therefore tell after you've let them self-fertilize.
Homozygous individuals have identical offspring for the trait when selfed, whereas selfing heterozygotes have a quarter of their offspring looking different for the trait.
In other words, you can't tell in the present generation who is heterozygous without molecular methods unavailable to Mendel. But if you are willing to self the individuals, save seeds, plant them, and track where they came from, you can figure out who was heterozygous/homozygous in the last generation.

So, does that mean that heterozygous pea plants (Tt) do not exist naturally? Again, I may be wrong.

Not for long.  Only half of the offspring of a heterogeneous organism will be heterozygotes if they self.

@swbarnes2 Heterogeneous plants will rapidly disappear when self-fertilization is the only reproductive path.  Crosses can happen in nature, though.


text:
Would muscles still twitch if the applied potential is reversed?
In Galvani's experiment with frog legs, he applies an electric potential across the muscles of a frog which causes the leg muscles to contract. What would happen if the polarity was reversed (I do not know the polarity he had originally used). Would the muscle still twitch?

Here the voltage is applied by using two different types of metals fused to each other

A nerve fiber is something like a cylindrical tube: it has walls (cell membrane) on all sides, and at rest is a bit negative inside, like this:

There are channels in the membrane that are sensitive to voltage. They are oriented with an inside-side and an outside-side, and move according to voltage such that they are closed when the inside is more negative than the outside. Most or all will be closed when the membrane potential is around -70 mV. If the voltage gets closer to equal, though, say -55 mV or more positive, the channels open, let more positive ions come in, and this current flows down the fiber depolarizing more membrane and opening more channels.
When you stimulate electrically with an external shock like in an experiment like this, the situation looks more like this:

You've added some huge voltage in some direction, here I've drawn it assuming the positive terminal is somewhere to the left and negative is somewhere to the right. For that brief time that the stimulus is on (it better be brief or you'll start cooking your tissue), you can just about ignore the little charge inside and outside the membrane that set the membrane voltage. All the membrane "sees" is your big stimulus.
For the membrane on the left, it's going to see a strongly negative voltage inside (towards the negative terminal) versus outside (towards the positive), and the voltage-gated sodium channels will stay closed. It doesn't really matter to the channels that this is extremely negative compared to normal rest: closed is closed.
For the membrane on the right, though, it doesn't have a negative voltage inside relative to outside anymore: the outside is facing the negative terminal, and the inside is facing the positive terminal. This is going to open some channels and let in some sodium ions.
A brief moment later, the external stimulus is gone, but those channels were opened and some ions were let in, making the inside less negative than it was, opening more channels, etc.
So, what if we flipped the polarity? Well, just flip the image, and apply the logic the same way. You can put the positive terminal into the page, too, and the negative outside (remember, we really have a cylinder here): as long as you make a voltage across the combined membrane of the fiber, you're going to stimulate a response. Even if your voltage source was at the top and bottom of the page, nerve fibers aren't perfectly straight, and anywhere that there is a bend will be susceptible to the shock (though not as sensitive as if the whole fiber was exposed across the potential gradient).
In practical, non-theoretical terms, sometimes the polarity you use does change the effectiveness a bit, but it's usually a subtle effect. I'll admit I'm not entirely sure what is physically behind this, but I think it has to do mostly with the electrode material and an inductance/capacitance that reduces the intensity of the voltage gradient depending on which way the stimulus is applied.
It's common in electrophysiology also to use biphasic square-wave pulses; in that case, the polarity reverses in the middle of the stimulation. This helps keep the electrodes in good shape and not depositing salts on them (the way you'd get in a battery-style chemical reaction), but it works just as well for stimulation.


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Size of Purussaurus, a giant Miocene crocodylian (2022)
What is the currently accepted size estimates (length and mass) of Purussurus barsiliensis? I quote the relevant part in Wikipedia.
From Wikipedia: https://en.wikipedia.org/wiki/Purussaurus

The skull length of the largest known individual of the type species, P. brasiliensis is 1,453 millimetres (57.2 in).[2] It has been estimated that P. brasiliensis reached about 10.3 metres (34 ft) in length, weighing about 5.16 metric tons (5.69 short tons).[2] Another estimate gave a larger size of 12.5 metres (41 ft) in length, ranging from 9.8–15.7 metres (32–52 ft), and 8 metric tons (8.8 short tons) in weight, ranging from 5.6–12.6 metric tons (6.2–13.9 short tons), with a mean daily food intake of 40.6 kilograms (90 lb).[3] It is also likely that Purussaurus reached only 10.9 metres (36 ft) long and 5.6 metric tons (6.2 short tons).[3] A 2022 study estimated a length of 7.6–9.2 metres (25–30 ft) and a mass of 2–6.2 metric tons (2.2–6.8 short tons) using a phylogenetic approach; and a length of 9.2–10 metres (30–33 ft) and mass of 3.9–4.9 metric tons (4.3–5.4 short tons) using a non-phylogenetic approach.[4]

Old estimates were up to 13 meters but more recent ones decreased it to 10.9 and the newest one from 2022 decreased even further:

A 2022 study estimated a length of 7.6–9.2 metres (25–30 ft) and a mass of 2–6.2 metric tons (2.2–6.8 short tons) using a phylogenetic approach; and a length of 9.2–10 metres (30–33 ft) and mass of 3.9–4.9 metric tons (4.3–5.4 short tons) using a non-phylogenetic approach.[4]

Reference to [4]: Paiva, Ana Laura S.; Godoy, Pedro L.; Souza, Ray B. B.; Klein, Wilfried; Hsiou, Annie S. (2022-08-13). "Body size estimation of Caimaninae specimens from the miocene of South America". Journal of South American Earth Sciences. 118: 103970. Bibcode:2022JSAES.11803970P. doi:10.1016/j.jsames.2022.103970. ISSN 0895-9811. S2CID 251560425.
https://www.sciencedirect.com/science/article/abs/pii/S0895981122002589
Since I don't have access to this paper I ask here few questions:

How did they estimate Purussaurus size?
What is a phylogenetic approach in the context of size estimates? I know phylogenetics is the study of evolutionary "family" trees and cladograms but how it affects size estimates?
What is a non-phylogenetic approach in the context of size estimates?
Why the two approaches yielded different estimations for Purussaurus?
Why the 2022 yielded a lower size estimate? Is it based on new fossils?
What are the scientifically accepted on Purussaurus size, considering the latest studies?

You are asking a lot of questions in one post, on a site that works better if you limit your question to one question - many of the answers to each of these questions will be full essay style answers in themselves. You will probably be better to break it up into several separate questions.

The estimates are based on 4 fossil skulls. As far as I can tell these are not new finds, the wiki link says the largest known skull as 1453 mm, and the largest of the 4 in the JSAES article is 1406 mm. This is probably the same skull measured with two slightly different techniques.
Animals don't just scale up linearly as they get bigger, even in the same species. Younger animals have different proportions from old ones, and different species have different proportions, of course. In particular, the ratio of body length / skull width is different. The 'phylogenetic approach' used in the article in question developed a statistical model for how this ratio varies in the family tree, then estimated the specific ratio for  Purussaurus. The details for this approach are given in another recent article, in case you're able to access that one.
The non-phylogenetic approach estimated the ratio with a linear regression of the full dataset, not weighting for relatedness.
As far as I can tell, the main reason the 2022 study gives smaller estimates of size is their exclusion of data from juveniles from the dataset used to estimate size from skull dimensions. I would have expected juveniles to have a larger head size relative to the rest of their body, but apparently that's not the case.


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Can a broken Swordfish's bill bone regrow?
The Broadbill Swordfish (Xiphias gladius) is characterized by a long bill or a "sword" which is an extension of its upper lip bone. The swordfish uses its sword in fighting, including attacking hard objects, which may cause it to break. Like all bones, it can heal after being broken but can it regrow back to full length?


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Why are scabies mites not found everywhere on the host?
The distribution of scabies lesions is not uniform. Sarcoptes Scabiei mites have an apparent preference for some places on human bodies.
They obviously prefer soft places, where they can burrow easily, but that's not the whole truth. For example, they generally avoid the face, even though there is plenty of soft skin to use. They burrow between fingers, but not on fingertips. They also completely ignore the scalp.
What factors are in play here? What makes mites so picky?