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How do burrowing animals get enough oxygen?
Tree borers make a small hole in a tree. At the end of it, they chew on wood to elongate the tunnel. They digest food and grow. All this takes oxygen.
The entrance to the tunnel is generally small. Some borers plug the entrance. Even without that, air circulation must be non-existent. How do they breathe?
It just seems off hand that tree borers might be an extreme case. But I have the same question about all kinds of burrows. How do worms do it? Rodents? Ants?
Do burrowers have any special behaviors or adaptations to help them cope?

Thank you all for your answers and comments. But I suspect there is more to it than that. I doubt enough oxygen diffuses down the length of the burrow to keep an insect alive under bark.
A tree died in my yard recently. The bark now comes off easily. When a tree is stressed or diseased, insect often attack. They finish off the tree if the original problem doesn't. Clearly that has happened here. They have eaten almost the entire layer under the bark. No wonder the bark is peeling off. There is almost nothing left of the wood it was attached to.

Most of the burrows are long and narrow. The insects carefully leave a small strip of wood between adjacent tunnels.
Here is a close up of two burrows from the bottom of the previous picture. It was taken before I pulled off all the bark. You can see they started at the bottom, and grew as they climbed.

Here is the bark that covered the burrows. When insects chew the wood, the wood does not disappear. At the bottom you can see a portion of the sawdust that packed the burrow. Most of it fell out when I peeled off the bark. I have seen other pieces of bark where it clearly filled the entire burrow.
At the top, you can see the holes where they dug out.

Now that the tree is dead, the bark is falling off. But it wasn't when they started. Trees do not make it easy for insects to eat them. I expect the bark was well attached. I doubt it was porous enough for air to penetrate. I doubt there was damage, such as the long crack you can see along one burrow.
Sometimes wood can be surprisingly porous. You can literally blow smoke lengthwise through a plank of red oak. But that is cut and dried wood. Live wood is full of sap.
I expect that insects do not make holes in the tunnel. They make it hard for predators to get it.
And yet enough oxygen does get in.

A year later, I still think about this off and on. While having a small mass reduces oxygen needs, oxygen is needed to chew a tunnel in wood and grow. I cannot believe that enough oxygen diffuses down a long narrow tunnel packed solid with sawdust.
Here is a photo from when I burned a log from the tree. You can see the surface is covered with burrows. There are also holes into the interior.

Here I have split the log. You can see the holes continue deep into the tree and lead to chambers. These burrows are almost entirely free of sawdust.

Here is a detail view.

I expect they are kept clear because insects need to crawl down them. One reason would be they need air.
Yet the surface burrows are packed. It seems likely an insect that can chew tunnels in wood can poke holes in bark. Does anyone know if they do this?
One last thought. I heat my home with wood. When I carry firewood home, I store it under this tree. People tell me you can transport insects in firewood...

Well it helps that insects and worms are cold-blooded, which means they have a slower metabolism and lower oxygen demand. How burrowing mammals handle air flow and oxygen could be different.

Oxygen is a small molecule, and can pass into that hole a million abreast!

@anongoodnurse We should look up the physical chemistry equations to estimate diffusion rate of O2 in and CO2 out. It might be interesting to see just how deep you have to go before it becomes a problem.

Many of these organisms are evolved to have low metabolic rates (oxygen consumption) and tolerance to high CO₂ levels. I know that this is the case with the naked mole rat.

As the body mass of an organism decreases, their volume decreases with direct proportionality, so their metabolic needs are also reduced. The result is that small organisms like worms and insects have a much larger surface area to body mass ratio, which lets them passively transfer oxygen into their bodies much more easily.
I am by no means an entomologist but most species of insects are able to exchange gases with the environment without any active respiration at all. Due to their small size, gases can passively diffuse through the trachea and spiracles directly to their internal fluids/muscles.
That said, insects simply have much much lower basal metabolic rates compared to even small vertebrates. Lower metabolic rates mean lower oxygen needs, so that's why its not uncommon to see insects surviving in cracks and crevices that don't have a lot of air circulation. The little air circulation that does occur in the temperatures that insects thrive in is more than enough to support their oxygen needs.


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Do babies resemble their father?
An often heard theory is that newborns and babies resemble the father more than the mother, a theory apparently ignited by a Nature paper by Christenfeld and Hill (1995). Figure 1 shows one of the tests images. His subjects had to guess which picture was the real dad and which was the real mom. It turned out that tested subjects were indeed significantly better at guessing dads than moms.

Test stimulus of Christenfeld and Hill (1995). Which one is dad: a, b, or c?
It is suggested that paternal matching of a baby promotes identification of the father with his offspring. In turn, self-identification in his kin would prevent the father from doing harm and stimulate protective paternal behavior. This then is hypothesized to increase survival rates of his offspring and et voila, a trait is born (pardon the pun) that makes baby paternal look-alikes fitter than those that don't. This hypothesis seems to have become part of parenting folklore.
However, a Scientific American paper by Matson (2011) contests this view and puts forward several primary research studies showing no preferred resemblance between mother or father, or even predominantly maternal features in babies.
Is the resemblance between father and baby fact or fiction?
References
- Christenfeld & Hill. Nature (1995); 378: 669
- Matson, Sci Am (2011) online article

Credits for this question go to @KoustavPal for his comment in Y-chromosome and phenotype

Most babies don't have a lot of (long) hair, while quite some of their mums will.

@AliceD: It applies to all infants, and to some extent to toddlers.  They resemble other children of similar age far more than adults.  I don't know why this should be, other than the workings of evolution.  Parents & other relative may say otherwise, but a) they're intimately familiar with their child; and b) there's a lot of social expectation/delusion going on.  It's de rigeur to say "Doesn't /he look just like dad?", though honesty would have you say that the infant looks more like an undifferentiated blob :-)

@AliceD: It'd be interesting to do a test (or perhaps find one that has been done).  Show an infant to an unrelated person for a short time, then ask the person to pick out that infant from a group.  I'd bet that results would be little better than chance.

I've heard an anecdotal claim that newborns resemble their fathers (which may not be the case in later life), and that it is an evolutionary adaptation to assure paternal recognition. This is usually corroborated with examples of animals killing progeny originating from other father, in order to give advantage to tehir own genotype. Is there any evidence for that?

I've heard of a study that found that individuals were more likely to tell the presumed father he looked like a child then the mother, and the mother's relatives were most likely to do so.  They theorized this was an adaptation to convince the male that he was the biological father to ensure he will continue to provide for the child.  As such it may be that paternity isn't that easy to tell when seeing a child, but everyone is still evolved to claim that the child looks like the presumed father.  Anecdotally I'm often told I look like kids I'm watching by those that presume I'm the father

@dsollen - interesting stuff. I've heard more of these stories/recollections but hard evidence I find hard to find. Hence the question. Thanks!

Resemblance between parent and offspring is certainly true. Some traits are more obvious than others, for example skin colour would be very obvious. As babies mature they will gradually begin to look more and more like their parents, the features of babies often change rapidly, for example pretty much every baby has a little nose, but by the time they are a fully grown adult it probably resembles that of one the parents fairly well.
More obvious example would be height - imagine being presented with a baby and two males who are identical in every way, except one is two metres tall, and the other is 1.5 metres tall. As a baby it would be very difficult to assign paternity on the criteria of height/size, but as the child develops it would likely become easier and easier.
The resemblance between parent and offspring relates to the heritability of a trait but, because there can be temporal variation within an individual, heritability can only be properly determined by measuring the traits in parent and offspring at the same time point (e.g. father's height at 18 years old and son's height at 18 years old), otherwise it will introduce noise in to the estimation, likely reducing the estimate of heritability (and specific to your question, making it harder to assign paternity based on phenotypic characters).
In the context of your question: Ability to assign paternity would depend on a) the resolution with which the traits available can be distinguished among all individuals in the test and b) the variance among all individuals in the test. E.g. If the only trait available was number of fingers per hand, and all potential fathers and the child had five then the lack of variance would make it impossible to use this as a criterion, while if both one of the potential fathers, and the focal baby, had six fingers on one hand then there would be pretty good clue.
Whether babies more resemble their fathers more than mothers (which I think is the true question you are asking here) there is evidence from Drosophila that within-sex heritability is much higher than cross-sex for lifespan and aging, i.e. the lifespan of males (females) far more resembles the lifespan of the father (mother) than the mother (father). This would suggest that there is a sex specific element. Meanwhile there is evidence of high cross-sex genetic correlations in humans preventing males and females from becoming more different (thus maintaining resemblance to opposite sex parents). I would think that resemblance to the father could offer some benefit in assigning paternity by care giving males (stopping males devoting resources to other males offspring) but some of the resemblance could be generated by genetic correlations to sex-specific genetic variance, for example, males (females) will look more like their father (mother) in many ways purely because they are of the same sex, so it will heavily depend on the traits you look at to judge resemblance.
For a related answer about heritability on biology SE see:
Is it possible for a child to grow taller than their tallest parent?

Thanks for your great answer. Are you saying that, in general, baby-boys resemble the mother and baby-girls their father?

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Why are human feces riskier than urine with regard to spreading diseases?
I came across a Wikipedia article titled "Urine diversion". It explains different ways, including using special diversionary toilets, and treating the collection of human feces and urine separately as opposed to just flushing everything down the toilet, maybe something most people in well developed countries are used to. These sentences caught my attention:

Separate treatment of the two types of waste is justified since urine is nearly sterile and low in pathogens, provided an individual is healthy.[3] This means that urine can be readily utilized as a fertilizer or discharged with less risk to community.[4]
Human feces, on the other hand are high in pathogens, including up to 120 viruses and need to be treated well before it can be safely used in agriculture.
Urine diversion - principle (Wikipedia article)

So urine is nearly sterile and low in pathogens, however it qualifies that statement with the condition that the person producing that urine is healthy. I assume when they compare this to feces they do so with all other things being equal (ie., provided an individual is healthy).
It claims that there may be up to 120 viruses in feces, as opposed to urine which it characterizes as being relatively low risk. I assume that feces are riskier in transmission of diseases because it naturally contains more pathogens, but it doesn't explain what these viruses or pathogens are. I was wondering if someone could elaborate on the risks of feces over urine, and also, notwithstanding that it says that urine is relatively low-risk, what health issues can be caused by reusing urine.

I like your question, but it seems a bit unfocused — I've made a suggested edit to the title that I hope better reflects the core of what you were asking.  If you approve of that change, I suggest editing the body to focus on that particular question.  I would then separate out the other question (about risks) into a new question.

@tyersome Thanks for the suggested edit. I edited the title and made the question about 50 words shorter. Hopefully it's more focused and clearer. Feel free to make any improvements/edits.

You're welcome — that is much better!

Many gut microbes are pathobionts or opportunistic pathogens — these organisms can be part of the normal gut flora (microbiome) of healthy individuals, but under the right (for them) circumstances (e.g.s: when introduced into the bloodstream by a cut, or due to immunosuppression) they can cause disease.1,2
As for urine, there are many fewer microbes present (>400 species and 109 to 1011 bacteria/g of contents in the large intestine vs. <100 species and on the order of 105 bacteria per mL of urine).3-5  Note, however, it is still far from sterile!5-7
You might also find the following of interest:
https://www.who.int/water_sanitation_health/dwq/iwachap5.pdf

References:
1: Hornef, M. (2015). Pathogens, commensal symbionts, and pathobionts: discovery and functional effects on the host. ILAR journal, 56(2), 159-162.

2: Price, L. B., Hungate, B. A., Koch, B. J., Davis, G. S., & Liu, C. M. (2017). Colonizing opportunistic pathogens (COPs): the beasts in all of us. PLoS pathogens, 13(8), e1006369.

3: Davis, C. P. (1996). Normal flora. In Medical Microbiology. 4th edition. University of Texas Medical Branch at Galveston.

4: Barnett, B. J., & Stephens, D. S. (1997). Urinary tract infection: an overview. The American journal of the medical sciences, 314(4), 245-249.

5: Lewis, D. A., Brown, R., Williams, J., White, P., Jacobson, S. K., Marchesi, J., & Drake, M. J. (2013). The human urinary microbiome; bacterial DNA in voided urine of asymptomatic adults. Frontiers in cellular and infection microbiology, 3, 41.

6: Hilt, E. E., McKinley, K., Pearce, M. M., Rosenfeld, A. B., Zilliox, M. J., Mueller, E. R., ... & Schreckenberger, P. C. (2014). Urine is not sterile: use of enhanced urine culture techniques to detect resident bacterial flora in the adult female bladder. Journal of clinical microbiology, 52(3), 871-876.

7: Whiteside, S. A., Razvi, H., Dave, S., Reid, G., & Burton, J. P. (2015). The microbiome of the urinary tract—a role beyond infection. Nature Reviews Urology, 12(2), 81.

It's not sterile, but it has far fewer microorganisms than nearly any other bodily fluid.

Urine is contaminated with skin bacteria and possibly something excreted from your blood. A very tiny fraction of urine is microbial.
Feces is basically a solid mass of undigested food, gut bacteria, and their byproducts. Many gut bacteria in particular have evolved in the conditions the human body produces so many can survive well in human tissue. about 30% of the solid weight of feces is bacteria or bacterial byproducts.
All things being equal, feces contains orders of magnitude more bacteria than urine by unit mass.

The kidney is essentially a filter which extracts dissolved waste products & water from the blood.  It basically allows only molecular-sized things to pass, otherwise it would continually leak blood cells.
The digestive tract OTOH is basically a tube.  Stuff gets mashed up at one end and ejected at the other.  Even fairly large objects can pass through unaltered.  So any bacteria that enter the tract can (if they can survive stomach acids &c) pass through the tract.  Moreover, many find the intestines a warm & comfortable place to live and reproduce.  Once the population has grown, some of it will be ejected with each bowel movement.  While most of the bacteria are harmless or even beneficial (see "gut microbiome" for more info), some, like some varieties of E. coli or the cholera bacterium, can cause serious illness.

Thanks, this addresses my query about the difference between feces and urine. I was just wondering, since you said that E. Coli or the cholera bacterium can be found in stool, does this mean that the person who passed the stool is infected with these bacteria? If that's the case, are we still talking about a "healthy individual"?

I can only speak to what I am familiar with, but I would assume that the large majority of those viruses are bacteriophages. These bacteriophages, or "phages" for short, are viruses that infect bacteria. I should mention that these phages are not human pathogens though. When we go out into the field to discover novel phages, we often go to wastewater treatment facilities to get a sample of raw sewage. These samples contain billions of phages, and after some treatment, you can use the sample and a stock culture of bacteria to propagate that phage. The pathogens are also most likely opportunistic pathogens, like staphylococcus epidermidis. These are bacteria that are a component of the normal human microbiome but may become pathogens in the right circumstances. You could also find clostridium bacteria in human feces and salmonella among many others. Also, if you are infected with an infectious agent, bodily fluids and stool will be a easy place to find them.

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Where different types of proteins can be found in food?
According to https://www.youtube.com/watch?v=KSKPgaSGSYA (created by one of the largest supermarket chains in the UK), different proteins have different roles in human body:

(Group 1) They’re like building blocks of you body, they help grow and repair your muscles, skin, hair, and nails.
(Group 2) They help protect you from getting ill by attacking germs of bacteria that might make you unwell.
(Group 3) They help you breathe by carrying oxygen around your body.

The following food is rich in proteins: meat (e.g. chicken, ham, beef), fish (e.g. salmon), eggs, tofu, lentils, beans, etc. but I couldn't find out which food is rich in which specific group of proteins, e.g. "eggs - Groups 1 & 3".

It does not really matter what “protein groups” are in a food, as during digestion they are broken down into smaller peptides. The main thing is to make sure you are getting the correct amount of the different amino acids for the body to then make those proteins you are describing.

Thank you for your answer! My question was purely out of scientific curiosity as I'm a quantum chemist and would love to learn more when it comes to nutrients. I'm trying to explain proteins to my little daughter.

@Hawkeye perhaps you should expand your comment into an answer.

I@WYSIWYG — wouldn't it be better if this question was migrated to a nuitrition/health list, as it is incredibly naive and, as pointed out, is  based on the misconception that the proteins in one's food preserve their functions after human consumption. The poster is as well reading her daughter the blurb on the back of cereal packets.

Possible duplicate of How a vegan can consume all the needed varied proteins / amino acids?

Having watched the video listed I see the confusion. Actually protein from different sources is interchangeable.
The video FIRST talks about what foods are high in protein like meat, fish, eggs and tofu
THEN about the different ways we use proteins in the body which it lists a few of not meant as a formalised 3 group system.
These two sections of information are meant separately. All the proteins are made up from only 20 discrete amino acid building block types (sometimes with a few modifications), when digested the body returns the proteins to their amino acids building blocks and reuses them. This effectively cycles left to right and back in the diagram below.

Amino Acid building block makeup is not identical between different foods but is usually roughly comparable. So all the sources of protein are interchangeable like a pile of 20 different colours of Lego brick, especially in a western diet it is very unlikely you would run short of any of them. The body uses spare amino acids as a food source and or can switch their types for reuse.
For more info and an interesting read on the amino acid make up of different foods have a look at the USDA database here https://ndb.nal.usda.gov/ndb/
And a more detailed look at the protein digestion itself in a good video "Protein Digestion and Absorption" by Armando Hasudungan https://www.youtube.com/watch?v=HL7-fYBtJ1Y

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Difference between AjuA and ajuA in biosynthesis
I cannot for the life of me figure this out. In the following figure, you can find ajuA and AjuA for example.
I do understand that the lower-case ones are the genes. The arrows indicate the direction of transcription. However, what is the difference to the upper-case AjuA? The text speaks of subunits. How are the two related? When do I speak of ajuA and when would I be talking about AjuA?
I would also be happy if you could point me in the direction of some literature about the basics of this kind of notation.
PS: I do understand the mechanisms of the biosynthetic route and am familiar with the different domains in the modules as well as PKS and NRPS modules.

It's just the nomenclature:

Bacteria: Gene symbols are typically composed of three lower-case, italicized letters that serve as an abbreviation of the process or pathway in which the gene product is involved (e.g., rpo genes encode RNA polymerase). To distinguish among different [but related genes], the abbreviation is followed by an upper-case letter (e.g., the rpoB gene encodes the β subunit of RNA polymerase). Protein symbols are not italicized, and the first letter is upper-case (e.g., RpoB).

[source]
You can check out the Wikipedia article on Gene Nomenclature for more information.

But what is the relationship between gene and protein symbols? Can they be used interchangeably? The paper talks about AjuK containing a module, while "genes ajuK and ajuL encode the first two biosynthetic proteins". Does that mean, the gene ajuK for example encodes the protein AjuK, which in turn does biosynthetic stuff?

@basseur "Does that mean, the gene ajuK for example encodes the protein AjuK, which in turn does biosynthetic stuff?" Without reading the paper, yes that's what it sounds like.


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Describing the location of the thalamic adhesion in simple English
I'm trying to describe the location of the thalamic adhesion as part of a personal project. I can't reference pictures, and I need to be as precise as possible without using convenient anatomical terms (e.g., dorsal, superior, etc.).
I have to use plain and simple English, with a consideration for people who speak English as a second language. My approach is to describe its location relative to more prominent and easily recognized body parts (e.g., bones and organs on the face or side of the head).
In every English construction I've tried, I first start by describing it as being located in the middle of the head. Then I describe its location in reference to common body parts on the transverse and coronal planes.
Most X-ray pictures I've found are only somewhat helpful to me here, because they're a sagittal cutaway of the head but they don't include the ear. Based on the X-rays, I'm confident that it's located at about the same level as the eyebrow ridge on the transverse plane.
The big problem I keep running into here, is that almost every drawing and rendering I find on the internet seems to put the thalamus in different locations on the coronal plane. This makes it pretty hard for me to find a precise coronal location for the thalamic adhesion.
In some of them it seems to be where the tragus of the ear meets the mandible. Some of them draw it further forward, more towards the mandible. In other illustrations it's drawn further back, almost directly above the helix of the ear.
I'm not a physician and I can't remember how many years ago it was that I took an anatomy and physiology class. So my question is, where is the thalamic adhesion actually located in the human body on the coronal plane? Is it in line with the tragus, or the ear canal, or some part of the mandible, or does it vary from person to person?

Here are two possible translations of the above into simple English constructions:

The "thalamic adhesion" is in the middle of the head, as high as the eyebrow ridge, and as far back as the ear canal
The "thalamic adhesion" is in the middle of the head, as high as the eyebrow ridge, and as far back as the temple

Secondary question: could anyone with a more expert knowledge of human anatomy suggest better constructions?


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Calculation of blood flow rate and pressure change in heart
Question: Blood flows from the left ventricle of the heart through the aortic valve and then to the aorta for system circulation. In a patient, the diameter of left ventricle outflow tract (LVOT) is found to be 1.6 cm, the aortic valve 8.0 mm and the aorta was 2.0 cm. Vitals of the patient indicate a 5.0 L/min cardiac output, a heart rate of 70 beats per minute and a duration of the ejection phase during each beat of 300 ms.

What is the volumetric flow rate during the ejection phase of the beat?
What is the pressure change over the valve?

You can assume the density of blood to be 1.06 g/cm3.
(answers: $Q$ = 0.24 L/s, $Δp31$ = -440 Pa)
For the first question, I tried to use the formula:
$Q = AV$
Where $Q$ = Flow rate, $A$ = cross-sectional area, and $V$ = velocity.
I calculated $A$ for LVOT, valve, aorta with $\pi r^2$, and calculated $V$ by dividing the diameter with 300 ms, and calculated $Q$ (with unit of L/s) by $AV$. Then I calculated the volumetric flow by adding each flow rate, but the result wasn't 0.24 L/s. I guess my approach is mistake, but I don't know how to solve it then.
For the second question, with bernoulli's equation, I tried to calculate the pressure change by $(0.5)*density*((V $of lvot$)^2-(V $of aorta$)^2)$, but the result wasn't -440 Pa. What are other things I should consider?

Is this possibly better suited for medical sciences? The question can be migrated if you want.

It's more of just a physics question; no biology or medicine is needed to answer.


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How do the lipid nanoparticles in the Pfizer and Moderna vaccines contain and release the mRNA payloads at the right time?
The engineering challenge with mRNA vaccines is that mRNA is fragile and degrades quickly. The solution, then, is to encapsulate the mRNA within lipid nanoparticles that carry the payload into cell.
However, this leads to another problem because mRNA and lipid molecules are both negative and will repel each other. In response, researchers developed cationic lipids that would bind the nucleic acids for delivery into acid. There was yet another problem, because testing found that these solutions were causing undesired side effects. The ultimate solution was to develop a lipid specially formulated to be neutral in at physiological pH, but cationic in acidic environments. This means that the lipid nanoparticles will be stable in blood or extracellular fluid, but once it is absorbed into the cell via receptor-mediated endocytosis, the acidic environment of the endosome will protonate the tertiary amine of the lipid into an ammonium cation, and the mRNA is released into the cell.
It's this last part which confuses me. Specifcally, why does protonating the amine to ammonia cause the mRNA to escape the nanoparticle? Shouldn't doing this cause the mRNA to be more strongly associated with the lipid, since cations and anions attract each other?


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Buffer Preparation Acetate pH 4.8 for alkaline lysis
This question might sound silly to many, but here we go.
Could someone please tell me the correct way to prepare 5M Potassium Acetate buffer pH 4.8 (3M w.r.t Potassium and 5M w.r.t. Acetate) for plasmid mini-prep?
Does the buffer need to be pH 4.8 or can you use a different pH?

Generally we look for evidence that you've made some attempt to answer the question yourself - have a look at Current Protocols, Cold Spring Harbor protocols or a copy of Molecular Cloning A Laboratory Manual, or even do a search for something along the lines of "prepare 5 M potassium acetate"

Hi, I am sorry that I wasn't being specific. I was wondering what the pH of the above mentioned solution should be? Mixing 60ml of 5M of KCH3COO- , 11.5 ml glacial acetic acid and 28.5 ml water results in a solution of pH 5.2. I am having problems with my plasmid preps and have read elsewhere that the pH of the said solution should be 4.8. I find it difficult to adjust pH of this solution to 4.8 as adding water to make up to the required volume resulted in pH going down from 4.8. Is it ok to use this solution with pH 5.2 or should the pH be strictly 4.8?

In that case you should dissolve the acetate and titrate pH with the acetic acid. The chemistry works by alkaline lysis, you then neutralize with acetate, so yes, you do need the pH to be correct otherwise you risk nicking the plasmids. Make sure you use fresh NaOH in the alkaline step - it does degrade with exposure to air when in solution.

The pH of the buffer should be at least close to 4.8 as the protocol relies on alkaline (NaOH) lysis of the bacteria, followed by neutralization of the alkali by addition of acid. If the pH is not correct you won't have enough acid to fully neutralize the alkali.
If you do not correctly or promptly (within the recommended 5 min) neutralize the NaOH, you risk damage to the plasmid by hydrolysis.  If your pH is not correct at either the alkaline step or the neutralization step then your protocol will not give good yields.
I find it best to neutralize within a couple of minutes of lysis, having determined that you have lysed as many bacteria as possible in this time. The best way to do this is ensure that you have fully resuspended the bacteria in the GET (resuspension) buffer with no lumps floating around, which means that you can add the neutralization shortly after adding the lysis solution and mixing.


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Identification of an animal building

I live in south-west Germany and today while mowing the lawn I discovered a strange structure. It looks like a molehill on which additional branches were placed. This kind of branches are easy to find in my garden, because they were removed from the trees by a strong wind. However, I have no explanation for this accumulation of branches on a small mound of earth. I have seen an animal that looked like a rat in the last few days.... But I think a rat would not build such a building? What kind of animal lives in my garden?
Additional Picture:

I would like to edit my question to provide the additional information requested by "tyersome":
There were high winds about two days before I discovered this structure. However, what is disconcerting to me is that there is obviously soil under the branches here, and the branches are conscientiously piled up.  The other branches are widely scattered in the garden. There have often been strong winds, but a similar structure has never been created before. I have been unable to detect any other signs of animal activity associated with this structure. I have left the structure undisturbed and have not investigated it further so far, as I believe it to be an animal burrow and I do not want to cause any disturbance to the inhabitants. Thus, I have not cut the grass around this structure, but have left it standing. Animals that can be observed in my garden are for example: pigeons, squirrels, other birds, mice.
Second Edit: There are trees nearby. Could it be a bird's nest that fell out of a tree? In addition: Is it safe for any occupants if I examine the structure more closely or turn it over?
UPDATE:
Day 2: Encouraged by your valuable comments, I examined the structure more closely today. Turning it over showed that it is probably, as you suspected, a bird's nest. There was also no entrance in the ground where the structure was located. I could not find any eggs or feathers. Therefore, the species of bird that built this nest remains unknown. The nest has a total diameter of 80 cm (31 inches). The diameter of the inner ("soft") nest is about 20 cm (8 inches). I didn't expect such a big bird's nest in my garden.

The nest may have fallen from this tree:

UPDATE (10. May 2021):
Since I had to assume that additional information would be needed to determine the species, I again examined the area extremely carefully today. In doing so, I was able to find eggshells that were located near the tree. I cannot say with certainty that these eggshells are directly related to the nest, but it is likely.

Then I researched, which bird eggs might correspond to these.
Based on the appearance of the eggs, I suspect the following species: The Common Blackbird (Turdus merula), the Eurasian Magpie (Pica pica) and the Eurasian Jay (Garrulus glandarius).
I have observed each of these species in my garden:
The Common Blackbird: very often,
the Eurasian Magpie: occasionally,
the Eurasian Jay: occasionally
Wikipedia states that the eggs of the Common Blackbird (Turdus merula) are  2.9 × 2.1 centimetres (1.14 × 0.93 in) in size.
The eggs of the Eurasian Magpie (Pica pica) "measure 32.9 mm × 23 mm (1.30 in × 0.91 in)".
However, I could not find any information about the size of Eurasian Jay eggs.
I would guess that the blackbird is too small to carry the large branches that are used in the base of the nest. I have also seen a blackbird's nest in a bush in my garden: I think the eggs were smaller and the nest was also made of much finer material.
So the Eurasian Magpie, as stated by "tyersome", is a very hot candidate for me. Can this be confirmed from the eggshells?

Welcome to Biology.SE! If possible please [edit] additional information into your post. Is there any chance this fell out of a tree or bush? You refer to branches being removed due to strong winds — did you have strong winds in the day(s) before you observed this structure? Can you see any other signs of animal activity around this — e.g.s droppings, feathers, hairs, other fibers? Finally, if you moved this structure did you find a hole underneath?

I, too, thought it looked like an upside-down bird's nest. If that were the case, there's no harm in examining it more closely; the possibilities are 1) there were only eggs (now smashed), 2) dead hatchlings, or 3) it was an empty/old nest. If anything existing had survived, you'd have seen parental bird activity (at least initially). This is assuming it's a bird's nest.

Possibly the least effective hedgehog of all time?

Great updates — 20 cm is quite big so I would guess this might be a largish corvid. One example (of many) would be the Eurasian magpie — the black-billed magpie of North America has a nest of similar size and construction. If you don't get an answer here you might try contacting local birding organizations. You probably need someone with local expertise. I also encourage you to note of what birds you are seeing and hearing — the builders could still be in your area.

Firstly, I thought that it was a Dusk-footed-rat from the image of the inverted, upside down nest. But, thanks for your updated clarification, I was able to Google Lens Search the images and here's what I found:- It is nothing to worry about, as the egg shells that you found are of House Sparrow's eggs. These birds can be commonly found and they usually make bigger nests (as you found in your garden).

Google lens doesn't know correct answers. That's a terrible way to try to identify anything with accuracy

You are crrect. That's why I rechecked Sparrow nests and eggs on Wikipedia and other sites then I gave you the answer.


text:
How to select a microscope for deep microscopy? Specifically for Deep Learning/ML model development
I am new to biology and microscopy. I am a data scientist. I am faced with a question on selecting a good microscope that enables automation of scanning the slides. And cellular object detection using deep learning.
Considering, my lack of background I am only looking at the resolution of scanned output and the ability of the microscope to provide images that can integrate with a workflow that I am about to develop. As one of the first applications, I am looking to detect sperm cells stained with Christmas Tree stain (head = red/pink, tail = yellow/green) using brightfield microscopy.
What are the other criteria I should worry about? What questions should a data scientist ask?
Any general advice will also be valuable.

Optics Optics Optics - there are a few questions you need to ask yourself and then supply in your question here, the first of which is "what are you looking for?" (cells? what sort?, stained (yes/no, what stain?) The second of which is "how am I looking for it?" This one might involve words like brightfield, fluorescence, confocal . Thirdly - how magnified? Stick to a quality brand (Nikon, Zeiss, Olympus) and you can't go really wrong, but be prepared to spend $100 K. Talk to your local sales reps!

The first application is the detection/localization of sperm cells using a Christmas tree stain. Brightfield, that is what the team I am working with mentioned. 40x.

There's already an automated platform out there for the use-case.  I've edited your question to include the information from your comment.

I would advise someone new to biology and microscopy to outsource this particular decision to someone who is not new to biology and microscopy: you need a collaborator. I realize this is probably what you intend to do by asking here, but I think you'll need more hands-on advice and guidance in this endeavor than you can get through StackExchange.

@BryanKrause I had just come to the same conclusion. I was going to start answering the question, but then realized that I would need to explain pretty much every part of the microscope and its purpose, which would take me far too long to write up, and be far too unwieldy to work in this format.

I do collaborate with Biology scientists. But I am the only one from a data science background. My question is more limited to the intersection between microscopy and data science. What questions should a data scientist ask when buying a microscope?

@kosmos: for list of information with possible questions to be induced see here. The questions you should be asking boil down to "what are the best things for my scenario?". This will include lenses, camera (CCD, colour/false colour), mounts, stage automation, illumination, image enhancements (DIC/Normarsky, Phase contrast, others), and even then it will come down to some opinion based things. You really really need to talk to an expert. I suspect that you will need mostly information about the camera data output and maybe drivers etc.

@kosmos I think the two sides of that question are best served by a conversation between A) You. You're the data scientist. You know what your processing pipeline is going to look like, so you have an idea what data you are going to need to input, and B) Someone who is an expert in microscopy. In particular, automated microscopy, which itself seems like a niche area to me. It's something where you're better off sitting down and having a back-and-forth conversation with someone who has the complementary experience to yours. Probably also a useful third party would be a sales rep.

@Bryan Krause thanks for your advice. There are biologists in my group but not experts in microscopy; not in an in-depth way.  We are actually talking to sales reps, but difficult to verify their claims. Considering a $100k budget, which is not small for the lab, I have a responsibility to ensure that the new microscope will enable data science applications such as sperm cell detection and potentially more.

@bob1 I have raised some questions with the sales rep and have requested a meeting with their technical team. My first question is the ability of the microscope/scanner to integrate with the pipeline. Particularly issues with proprietary formats. Regarding image quality, my assumption is that if the human eye can identify it, the machine could be trained to do that with sufficient data.  I find that object detection in microscope images has a challenge that cells are too small amidst a lot of background objects. Considering this and in general, what would you comment on this assumption?

I think you really need to provide more information on just what you're attempting to do.  Just what do you mean by automating the scanning of slides?  Do you have humans preparing the slides and putting them on the microscope?  Or do you want an automated pipeline to do some or all of that?  For the former,  you could probably (though I'm no expert, by any means) use a much less expensive microscope with a high resolution USB camera.  Especially if you're still in a development phase, where your main concern is getting the software to work.

Not sure this is answerable here. I suggest trying to get a demo or better yet tell the sales rep that you need to "test drive" their product for x days — where x is significantly more time than your worst case estimate for how long a test run would take. Maybe invoke/invent a manager/supervisor/bean counter who is skeptical and thinks a competitors cheaper scope would be more than sufficient for your needs?  Probably works even better if you actually have specifics about alternatives ...

For detecting objects like cells, you need a good enough spatial resolution that comes from the choice of objective (the higher the NA, the better the resolution).  No need to worry too much about that, though, if your object is larger than ~1 um. You also need a high dynamic range from the detector, preferably 16-bit or higher, and to make sure that the experimenter will use it. Proprietary software is a problem, mostly if you want to do real-time detection. You can always build your microscope to custom specifications but that requires expertise.

@vkehayas Thanks for your comment. It is not real-time detection.

@tyersome These microscopes are quite expensive and getting one for trial is a bit of a problem, especially if they need to come from another country.

@kosmos In my experience, the more expensive something is the easier it is to get one for a trial. The sales folk know you are making a big investment and are willing to do anything to make it happen. You say you have biologists but not microscopy experts in your group. Biologists aren't good enough - get a microscopy expert, too. For things like proprietary format...that probably comes from the camera rather than microscope. Every camera I've used could output in some sort of bitmap format. Video was more problematic.

@kosmos if you are doing single image, most software(!) can output in tif format, but this might not be enough for you, depending on your pipeline. I've had good use with Spot cameras, they have excellent resolution, great optics and good software. They also have a development kit for integration with custom software, which might be useful for you.

@kosmos regarding your questions in comment - yes, and no. I'm no expert in machine learning or anything, but the trained human eye is very very good at picking up patterns. If the cells are too small cf. other objects, increase the magnification. Optical magnification is better than digital, for reasons that should be clear to you.

@bob1 thanks for your comments.

@kosmos - ignore my comments re spot cameras, they were good about 10 years ago, looks like they haven't increased the pixel resolution (5-12 MP) since then. This may not be a problem for you though, and most microscope cameras are in that range anyway. Another option is to get adapters for a D-SLR camera.

Are automatic sliders available as extensions to microscopes? By automatic slider, I am looking at a process where a few hundred slides are prepared and placed on the slider and it automatically pushes it to the microscope. The microscope does the scanning. The scanner/camera captures the image and stores it on to the cloud or on-premise storage. These images are then either seen by an expert or processed by automated software. I see there are cameras that could be added to microscopes. But looking for automatic sliders as well which work with the cameras.


text:
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.


text:
Pathological mechanisms in asthma
What is known about the precise molecular mechanisms involved in asthma?  For instance, is it known why the bronchi tissue alteration cannot be reversed?  Were any new mechanisms discovered in the last decade or so?

I don't know the answer myself off-hand, but this seems like a rather basic question, so you should probably look it up somewhere else (such as Wikipedia, or a medical textbook) rather than asking people to explain it here.  On this site you're only supposed to ask for information that you couldn't easily get ready-made.  If you have in fact looked it up already without success, you'll get a better reception if you say so and tell them something about where you've already looked.

Yes I understand, but my hope was that someone who knows well that topic could give me the great lines, so that I could find by myself the details more easily.


text:
What is this black worm-like creature with a yellow underbelly
This was found in the Malay Peninsula around noon. Unfortunately it was doused in hot water by my grandmother.
Image below is after having been killed by hot water.

It is about 1.5cm-ish long. It was curled up for some time but it started to move when it was exposed to sunlight(before it was killed). According to my grandmother, she had seen these long ago and they would stick on trees and foam up when touched. It didn't seem to foam up after it was killed though.
What species is this and is it endangered?
Edit : I'm very sure it is a type of chiton now and that it has already been doccumented. I'm saying this because I saw a video on YouTube with an extremely similar creature. Although I am still not sure what species it is.

Any chance you have a photo of the other side. It looks to be a slug or perhaps a flatworm of some sort.

No, I did not take a picture of the other side unfortunately.


text:
Cumulative synaptic transmission delay
Assume the following:

there are at least 10^11 neurons in the human brain
there are approximately 10^14 synaptic connections in the human brain (because on average each neuron gets inputs from approximately 1000 other neurons)
synaptic delay is approximately 1-2ms (but for the sake of it we can also assume an order of magnitude less, so 0.1ms)

A problem that revealed itself to me:
It would "only" take 1000 (or 10000 if we use 0.1ms as synaptic delay) serial synaptic connections in order to generate a lag of 1s. Considering that there are 10^11 neurons in the brain, the number of neurons you need  firing sequentially in order to generate significant lag (>1s) seems tiny compared to the overall amount of neurons in the brain.
My questions:
Are there series of neurons (firing from the time of input to the time after processing) greater than 1000 (or 10000)?
How are there series (not) greater than 1000 (or 10000) neurons?
P.S. I'm talking about chemical synapses which I assume to make up the bulk of neural transmission in this case

Welcome to the site.  Where did you get your information? Particularly, where did you get the idea that all the neurons in the brain are connected in series?  How on earth would the brain process information at all if there weren't millions of different short pathways that the "signal" could take?

The information is from the Springer Handbook of Bio- and Neuroinformatics. I do not (!) think that the neurons in the brain are all connected in series. I'm just asking how come that there isn't significant lag as it would take only a series of 1000 or 10000 neurons in order to create a lag of 1s. My question is more like: how aren't there series of neurons that are greater than 1000 or 10000?
I basically have an idea how to answer my question but I want to compare it with what people on here say. I'm not a neuroscientist and my reasoning doesn't suffice for me personally.

To clarify even further: I'm particularly interested in:
Are there series of neurons greater than 1000 (or 10000) Yes or No?
And subsequently: how are there series (not) greater than 1000 (or 10000) neurons?

Okay I'm starting to catch your question now; it would be great to [edit] this information into your question for clarity.  Not an answer here, so I'm just putting it as a comment, but there are definitely large variations in the number of neurons in a given circuit - think of reflex arcs which respond extremely quckly - there can be as few as 3 neurons implicated. Once you get into higher processsing estimating how many neurons are in series could be exceedingly complicated, since you (almost) never have isolated neuron-neuron connections (look up convergence and divergence).

...rarely would it be the effect of one neuron alone causing another neuron to fire; the whole idea behind processing is not to send information through the most processing centers possible; it's to take information from the most sources possible and integrate it into a single response.  That said, some processing does take much longer than 1s if it has to go through lots of circuitry, sometimes multiple times - think of doing mental arithmetic - you have to store numbers and recover them, process them, etc.

...and some series of neurons are indefinitely long - for instance, you can close your eyes and start imagining a story, and technically there is no limit to the number of neurons in circuit which you can make fire.  You could spend two hours imagining a story (or whatever) and there would be way more than 10000 neurons that fired dependant on others.  Anyway not an answer; just food for thought which will hopefully help you clarify the question a bit

Lags of 200-600 ms are pretty common. Have you ever read studies that measure reaction times? That said, total synaptic delays are typically much longer than 0.1 ms (that number is just for one step in the process). 1-10 ms is a better lower estimate.

I'm aware of these lags (200ms-600ms), I also encountered them when looking for an answer to my question. However, let's say this roughly equals 200-600 Neurons or synaptic connections, I'm still astounded it's that few. I would have expected it (sequential neurons) to be orders of magnitude higher and I'm asking myself how it is the way it is.

The brain is massively parallel. There are a lot of recurrent connections and feedback loops that are important for setting expectations, building a model of the world, and learning, but the pathway for stimulus to response is quite simple (see the diagrams in Grossberg & Pilly, and the latency profile in Schmolesky et al). It has to be, because if you are slow you die.
For example, a commonly used psychophysical task is to detect a motion stimulus and response with a saccade (eye movement). For this path you have a couple synapses in the retina, then the LGN, V1, MT, LIP, FEF, then a couple synapses in the brainstem and the muscle. For some of the cortical stops you might have responses that themselves depend on multiple synaptic steps. So let's say on the order of 10-30 synapses for that sort of rapid task with a response within a couple hundred ms. A good chunk of that is actually phototransduction in the retina which is quite slow (which is why you don't see still images a 24-30 fps movie).

DiCarlo, J. J., & Maunsell, J. H. (2005). Using neuronal latency to determine sensory–motor processing pathways in reaction time tasks. Journal of neurophysiology, 93(5), 2974-2986.
Gold, J. I., & Shadlen, M. N. (2007). The neural basis of decision making. Annu. Rev. Neurosci., 30, 535-574.
Grossberg, S., & Pilly, P. K. (2008). Temporal dynamics of decision-making during motion perception in the visual cortex. Vision research, 48(12), 1345-1373.
Schmolesky, M. T., Wang, Y., Hanes, D. P., Thompson, K. G., Leutgeb, S., Schall, J. D., & Leventhal, A. G. (1998). Signal timing across the macaque visual system. Journal of neurophysiology, 79(6), 3272-3278.

Parallelity indeed solves the problem of massive difference in magnitude between overall neuron count in the brain and serial neuron count from input to result of process for me. However, do you have more insight or a source about counts of serial neurons? Your answer probably is enough to resolve my question, I'm just dumbfounded why neural transmission is so slow and long serial neural transmission has the potential to be excruciatingly slow. Our brains are amazing but a complex instruction in Assembly like ldw (loading a content) takes only 100 ns with simpler instructions being much faster

Brains are biology. I would recommend learning how they work from the ground up (get a neuroscience textbook) rather than trying to understand them like you would silico computing. The brain-computer analogy breaks down really fast.


text:
Can insects be killed by intense visible light?
Some mosquitoes and flies were sitting on the white ceiling. I took a very powerful flashlight to try and daze and then vacuum them.
When I put the flashlight's head against the ceiling (with a mosquito between the light and the ceiling), I heard buzzing for two seconds... then it stopped. I removed the light, and, to my utter amazement, the mosquito was dead! Tried it several times on other small flying insects (including medium-sized flies) and not a single one of them could resist my light saber! :)
Could they really be killed by intense visible light (concentrated and reflected by the ceiling)?.. Note that the flashlight is not getting hot so it wasn't about heat (I can put it against my arm and it feels just barely warm).
EDIT
I would have to disagree with the comments.

First, why the downvotes?.. :)
NO, the insects are not being crushed by the torch. There's a lot of space between the torch's crown and the ceiling. As a proof — small mosquitoes and flies are quickly dead but a big fat disgusting fly was just buzzing around and then flew away!
NO, it's not the intense heat that kills them... at least not in contact form. Like I said, I can easily put the torch's head to my palm and it feels barely lukewarm.
Sentences like "skin can't be broken by light from a torch" or "insects are flying around in full sun and don't drop dead" are not substantiated, they're just some random opinion from Internet. For the contrast, here's a scientific article: https://doi.org/10.1038/srep07383
To quote:

Our findings suggest that highly toxic wavelengths of visible light
are species-specific in insects and that shorter wavelengths are not
always more toxic. For some animals, such as insects, blue light is
more harmful than UV light.

Granted they irradiated the insects for hours or days, but the point is that visible light can be very harmful for some insects (including, specifically, Culex pipiens molestus!)

Yes, the Sun is a very powerful source of light, but it's not concentrated. Like @canadianer said, the intensity is very high if all the light from a torch is forced onto a small space... and this is precisely what's happening (I specifically mentioned "white ceiling" because it causes all the light to bounce back on the insect!) When the torch is nearing the ceiling, the reflection gets so bright that it's very painful to stare at it (more painful than staring at the Sun)!

Ability or inability of a torch to burn skin has nothing to do with lethality. There might be many mechanisms behind this effect; I suspect their ganglion is overloaded by the intense light and it switches itself off!

And, finally, "common-sense logical answers" such as "insects fly all day in the sunlight and don't die" is not something I'd expect on SE. If you think that my conjecture is wrong, I'm fine with that, provided that you quote articles and list their DOI's.

I beg to differ @jakebeal. There're a lot of space between flashlight's emitter and the ceiling, the insects are buzzing and moving around. Moreover, they're not crushed, they look intact (even medium-sized flies, mosquitoes are much smaller but still they dropped dead!)

That laser weapon is physically damaging the insects @jakebeal; agreed, a handheld torch won't be powerful enough to do that. However, I was thinking that intense light simply overloads their CNS, it short-circuits and they drop dead! :)

@Alexander: Yet it's common to see mosquitos & flies flying around in broad daylight, which is much more intense than any flashlight I've heard of.

@jamesqf If you have a 1000 lumen flashlight held close enough to a surface such that it illuminates 0.001 square metres (about 3cm x 3 cm), the light intensity on that surface would be 1,000,000 lux. According to Wikipedia, that is about 10 times more intense than “bright sunlight”.

include the model of the flashlight and the distance and this can be answered to your satisfaction, otherwise all that is possible is just anecdotal conjecture.

Did you verify your insects were dead rather than momentarily blinded or confused into immobility?  This could make for a popular video, provided the flashlight doesn't kill the camera. :)

Without knowing the specifications of your flashlight (other than "very powerful") it is difficult to answer this question, however there is one common feature of all forms of lighting; heat output.
Most of the output of flashlights (including LED) is in the form of heat - more than 50% in the case of LEDs, greater amounts for incandescent bulbs (>95% IIRC). I've found a little information on this without diving into the literature too much.

Although LEDs are cool to the touch, within the devices themselves, there is plenty of unwanted heat. This heat comes from the inefficiency of the semiconductors that generate the light. The radiant efficiency (total optical output power divided by total electrical input power) of LEDs is typically between 5 and 40%, meaning that 60-95% of the input power is lost as heat.

I don't know how much of the heat is radiated out the front of the flashlight as this will depend on the flashlight design.
It's likely that you just got the insects heated to a point where they died from heat.

Why the downvote - it is the correct answer, nothing to do with biology or "white" light output.

For what it’s worth, I have an LED flashlight the manufacturer claims is 1800 lumens and I can’t hold it next to my skin for more than a few seconds. Shining it at my skin from an inch or two away and it still becomes too hot in 5-10 seconds.

@canadianer - I've had a minor burn (sore and red) on my leg from the LED flash/flashlight on my phone camera that was on in my pocket and I was unaware - that's going through the pocket material, so I am not surprised that an 1800 lumen one is hot to the touch.

I didn't downvote @bob1 :) Hmmm maybe... meaning that the insects are not heated directly from the contact with the bulb (because I can easily touch and hold it), but maybe the light heats them up instead... My torch is 1600 or 1800 lumens but it's a diving torch with a very effective radiator, so it gets hot only after a couple of minutes. When I switch it on, it's cold to the touch.

downvoted because it is not "likely" at all, you would at least have to show the heat output is at a dangerous level which is difficult without knowing the intensity, distance, and exposure time.

I think what is missing here is the proper experimental procedure, where all the effects, potentially harmful to insects (heat, physical pressure, etc.) are controlled. Think about making a movie of your experiment, doing it in different places and on different days. As now the discussion is meaningless.
Designing such an experiment could be an interesting question in itself, although I am not sure whether it is more appropriate for this forum or for Cross-Validated (statistics).
Note also that visible means a particular frequency range - the one perceived by our eyes. The frequency by itself is not harmful - what may cause harm is heating due to the energy absorbed with the light (microwave, infrared and ultraviolet radiation are known to heat not less than the one in visible range). Energy transmission with incoherent light (as opposed to the coherent laser light) is very inefficient, so it is unlikely to be the cause... yet, scientifically one cannot make any conclusions on the basis of the evidence presented so far.

The first experiment could be to simply use the torch while switched off. Who knows, maybe it's simpy the intense tries of the insects to flee - they might literally be "hitting their heads" on the glass and stunning themselves due to impact-

actually all you need to do is show insects survive higher intensity light just fine, which you can do just by knowing the flashlight involved.

@AnoE Is an insect actually capable of stunning itself?

@John It depends on the question one poses in the experiment: if one wants to test whether it is the  that flashlight kills the insects, than this could be the first step (because, if it can kill the insects, it will still remain to prove that this is the case). If one wants to clarify why they die (and whether they die), it might exlude the flashlight hypothesis.

@RogerVadim, that's the question. The scale of "physical impact" has two extremes: 1) no impact, and 2) the insect hitting my car's windshield at 200km/h. We know what the latter does to the insect. So somewhere between zero and 200 km/h there is a point where the insect will be almost, but not quite dead.  I have no idea whether insects can reach this kind of kinetic energy by hectically buzzing around in an extremely constrained space, but generally nothing surprises me anymore when hearing this kind of stuff... this would explain OP's question if it could be demonstrated in experiment.

@AnoE Another possibility is that the insects in question are not dead, but merely disoriented/paralized.

No, if the flashlight cannot harm your skin its is not killing an insect. In the absolute best case scenario you dazzled the insect temporarily in the same way intense light can dazzle you. lasers have been used to kill insects and they are quite intense, low intensity lasers far more powerful than your flashlight are used to identify insects and they do not kill them. Can you kill insects with intense visible light, absolutely, but your flashlight is nowhere near powerful enough.

"if the flashlight cannot burn your skin its is not killing an insect". Is it necessarily the case that insects require more energy to be killed that human skin does to be burned? I think some kind of logic or reference would be good.

@user438383 both are made of the same material, mostly water, and sunlight can burn human skin yet insects survive it just fine. lasers which can kill insects are quite painful on skin. keep in mind sunlight can burn human skin but will not kill most insects.

Like I said in comments, I don't believe either heat or the light itself is physically damaging the insects or their chitin carapace. In my opinion their brains are overloaded by the light...

@Alexander how do you think light can overload a brain? you have dazzling which I already mentioned, is temporary, and is not lethal. If your propose some other effect you would have to demonstrate such an effect even exists. And again sunlight is brighter and does not "overload" anything.

Hey @John, well it's called Sensory Overload in humans... loud sound, bright light or other stimuli can cause serious adverse reactions, even though they don't cause physical harm. I guess nobody died from it, but insects are very different from us...

@John, like some people said, the sunlight is diffused but torchlight is very concentrated and reflected from the white ceiling. Anyway, I'm not trying to prove anything, I just made an observation and asked a question and am addressing "obvious" answers such as "insects are crushed by the torch" :)

you said it yourself sensory overload (dazzling) does not cause physical harm. Also I don't think you understand how light intensity works as how diffuse the light is means nothing if the light intensity is higher if anything that means the insect is receiving even more energy from sunlight.


text:
Would it be possible to sequence human DNA from wastewater treatment plants?
I know that there are some efforts from scientists working with bladder cancer to perform  liquid biopsy from urine for detection and monitoring of the cancer such as this one.
I've been searching online - but the "wastewater" search term gives me tons of hits about "microbiome" and several types of pathogens.
But I'm really interested in sequencing the sewage to find and analyze human DNA. I have been wondering about liquid biopsies to get an estimate of the cancer status of a population.
I immediately think of several problems:
1. Technical Feasibility

Would we expect to find enough human nuclear DNA within the samples (taken across several days)?

2. Law and Bioethics

Is it legal in the US or European Union?
It could be difficult to get informed consent of all people who are connected to a certain sewage system. You may get informed consent from all residents but what about visitors?
It could be difficult to opt out of such study the
wastewater without a) moving to a different place b) using a septic tank.
I only found this elaborating other privacy issues: its just about Cov-19 and drug testing; both of which are not able to identify individuals at a genomic level.

I'd appreciate your experience and thoughts and references!
[Edit] as suggested in the comments: I have posted part 2 of the question to the law stack exchange here

Given the availability of sets of human reference-haplotypes I think it should theoretically be possible to gather some additional information to common mutations like SNPs even from sub-optimal waste-water. Just you have to be aware how incredibly polluted human DNA would be by incomparably higher DNA-concentrations from other species like microorganisms. Still, as it is possible to identify the whole range of micro-organisms from soil-samples using NGS, one can easily evaluate whether sequenced stretches are uniquely human. Anyways, why not directly get high-quality human DNA samples?

I once met a lady working at a waste water plant and she told me about the different samples they take. I found it fascinating and wondered about other applications that would only consider human DNA in particular as acquisition of participants for a study  is probably one of the bottlenecks. The question became 'hot' for me now as I'm doing an applied genomics course where we must invent a project with the requirement to do "very smart" and cheap sampling (his words). The project must be feasible and we are required to calculate a budget. --> why would not use high-quality human DNA :)

The U.K. tests wastewater for COVID currently https://post.parliament.uk/monitoring-wastewater-for-covid-19/

@Tim That is a totally different story as this is not looking into human DNA which can identify people... Thats the most personal data the any of us have to offer. Given you could make a Data Base of this human DNA. There are many ways this data could be matched to a region/community (where you poop) and to a network you belong to, possibly identifying you. That makes labeled DNA data. Given your computer science back ground I'm sure you can see why this is a totally different story and ethically concerning. Downstream one could even identify your relatives...

@ilamengl The bit about the practicalities of DNA sequencing are on topic. However, the edits which ask for a discussion on the law/ethics side of this are off-topic here. It also reads a bit like a discussion question rather than one with a specific, objective answer.

@user438383 they are 2 yes no questions in the post: The concrete answer to question is 1: yes it is legal - 2: No it is illegal. Question 2 can be answered as follows: 1: yes it is possible (see answer below) or  2: no its not

@ilamengl Either way, questions about the law and ethics are off-topic, and so is having multiple questions. Stick with the original bit about the scientific aspect of sequencing from waste-water and if you are interested in the legal aspect, maybe ask a question in https://law.stackexchange.com/

@user438383 given that people who have written proposals for scientific project had to think about those things and ask their local bioethics commission I feel like I got more chances here than in law...

@ilamengl Any "philosophical or ethical questions related to biology" are explicitly off-topic here, per the site help page.

As it turns out, you are not the first to have this idea. Prior research claims that it is, at least in principle, possible to conduct epidemiology of cancer by sequencing on wastewater DNA for biomarkers.
The ethics of this approach also need not be problematic because narrowly targeted sequence amplification focused on biomarkers like SNPs will not typically produce enough information to identify an individual.
The practicality of this approach is a major open question, however, given the small and variable nature of the signal.

Grate idea and grate paper -They are however using mtDNA. I am still wondering about the first part of my question is is ethically and legally possible to extract human cellular DNA.

If you can get mtDNA, then in principle you can get other DNA as well. The key issue remains signal levels.

@ilamengl a sewer grate? Sorry, couldn't resist.

@BolucPapuccuoglu LOL a Freudian slip.

@jakebeal thanks for answering the technical part. About the other part of the question: are you aware of any US or European laws that would prevent scientists from sequencing human DNA from the sewer without consent given it would be hard to get consent?

@ilamengl that might be a better questions for [law.se]

As already hinted in the answer by @jakebeal, the low concentration (high dilution) of human DNA in waste water would mean that we need huge sequencing depth in order to detect something. This is not the case when dealing with urine or feces. This is also not the case when sequencing bacteria in waste water, since we are dealing then with the strains that can live and multiply in water, i.e., they are present in important quantities. Moreover, we are interested in them only when they are present in high quantities, when they are potentially harmful.

The research group of the paper jakebeal linked used filters -> less water more cells...

@ilamengl it seems that their main achievement is precisely overcoming the problem that I described in my answer. It is a strong claim - if this is really the case, we might expect this done commercially in a few years. Note that they didn't do sequencing, and that they look only at mitochontdrial DNA (a small part of the DNA contained in a human cell).

@ilamengl it is human mtDNA. I think the catch is that they have a library allowing to amplify mtDNA. I am not sure, whether the same can be done for all of the nuclear DNA. If not, you have to shotgun sequence everything that is found in the water, which is the premise of my answer.

Again, I am aware that it is human mtDNA because I've actually read the article ;p but we both know that we cant find the same kind of info as within the nuclear portion of human DNA

@ilamengl if one is looking for a specific biomarker (e.g., a specific type of cancer) one is concerned only with this DNA fragment. I don't know what is special about mtDNA in this respect.

What if I wanted to find novel SNPs? And maybe their distribution within the population?

@ilamengl in most cases one can sample such data in less complicated ways.

Reputedly Osama bin Laden was tracked this way
... according to Water and Wastes Digest citing Time Magazine, but few of the details are known.  Given that 10 milligrams of feces is sufficient to amplify desired markers from DNA, you would need only consider the number of people living "upstream" to determine how much material you should need to collect overall, and how many sequences you would need to run to find the right one.  Maybe with some clever efforts to separate material into microwells you might be able to isolate one or more cells from the target of the search away from all others.


text:
How to test whether correlation of couples phenotypes is due to assortative mating or environment?
A few phenotypes are easier to pinpoint as assortative mating (height for example), But other phenotypes such as vitamin D, weight, etc could be a combination of shared environment and assortative mating. How could I disentangle those?
One idea was to compare against shared genetic variants associated to those traits. If couples also share these variants it is more likely to be AM rather than environment.
Do you have any other ideas? Unfortunately I don't have longitudinal data. And I only have data from mating pairs, not from offspring.
I am working with exome sequencing and genotype data. I have several phenotypes (anthropometric features, molecular traits, ICD10 codes).

Please give us a bit more detail. Should we assume you are working with human data? What kind of data do you actually have? Sequence? Variants? Expression? RNASeq? Phenotypical data? Family history? Clinical history?

I added some more details. I have exome+genotyping, phenotypical data, ICD10 codes.

I feel like you will have to account for population structure to say much about assortative mating. I'm not an expert in the field but I'd suggest reading up on what others have done, see e.g. https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1006655

I'm working with europeans with white ancestry only, so population structure shouldn't be much of an issue.

This strikes me as a question that might be able to be answerable for model organisms like drosophila, but is hopeless in humans. What do  you mean by a "vitamin D" phenotype? Can humans even observe it? "If couples also share these variants it is more likely to be AM rather than environment." Not true! If the couples also all share the same or nearly the same environment, then the genetic variants are completely uninformative. For example, my wife and I both have blue eyes, but that has nothing to with the fact that we both get up at 7am, that's because we share an alarm clock.

Vitamin D levels in blood seems to be a highly correlated phenotype in my set of couples when compared to random. When I meant genetic variants I meant the variants relevant to the phenotype. So for example, SNPs relevant for height should be highly correlated among tall couples. But SNPs relevant for circadian rhythm(?) as you mention may not. As the environment could have a bigger impact than genetic predisposition.

"Vitamin D levels in blood seems to be a highly correlated phenotype" ok, but for assortive mating to be happening the couples would need have some sense of whether a potential mate matches the desired level of Vitamin D. How on earth would that be happening? Isn't measuring genetic variants associated with the correlated phenotype just re-measuring the correlation? On top of all that you have a multiple testing problem: if you start with a laundry list of possible phenotypes and then go looking for weird correlations you are almost certainly going to find them but mostly by chance.

My sense is that you could really only answer a question this with a multi-generational program with a model organism.

Yes I have no idea why they're so highly correlated. Perhaps it is another phenotype (more visible) related to that one that is driving the signal. I believe I have enough sample size for multiple testing not to be a problem. But I'm still not sure how to explain this. I'm a bioinformatician, so a model organism wouldn't be suitable for me. I guess given I don't have before mating and after mating data, I would be better off doing simulations. I just have to figure out the correct way to do it.


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What is the density of nacre?
What is the physical density of nacre/mother-of-pearl? I can't find an authoritative figure for it.

According to this article, nacre is composed of aragonite ($\mathrm{CaCO}_3$) and organic matrix (chitin, proteins ...) with a mass ratio $95 \% : 5 \%$. In the supplementary file, they provide densities for each component, namely the density of aragonite $\rho_a=2.95 \; \mathrm{g/cm}^3$ and the density of a generic protein $\rho_p=1.35 \; \mathrm{g/cm}^3$. From this data, we can estimate the density of nacre:
$$\rho_n \approx \frac{1}{\frac{0.95}{\rho_a} + \frac{0.05}{\rho_p}} = 2.78\; \mathrm{g/cm}^3.$$


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Appropriate cell lines to study depression
Short introduction
I'm studying depression from a biochemical point of view. My interest lies in the study of protein biomarkers and I was wondering which cell lines may be appropriate for this purpose. It is not important that the biomarkers occur in easily accessible tissue or matrices, as this is not the exploration of a new diagnostic method. Rather, it is important to obtain the most accurate information possible about protein expression in the neuronal environment, where depression is supposed to occur.
In my literature research, I came across two quite commonly used cell lines, both of which have their advantages and disadvantages. Let's have a look at them:
LCL cells
LCL cells are produced by infecting lymphocytes with the Epstein-Barr virus.
Advantages:

They are relatively easy to have produced individually, so it is perfectly possible to get cell lines from patients with a diagnosed depression and from patients that are very unlikely to be depressed.
They were already used for studying depression-related topics [1, 2]

Disadvantages:

LCL cells are not neuronal cells, which means that they might (and probably will) express a different set of proteins than neuronal cells.

SH-SY5Y cells
SH-SY5Y cells were subcloned from a cell line that was isolated from a bone marrow biopsy taken from a four-year-old female with neuroblastoma. The subclones were chosen such that individual cells with neuron-like characteristics were selected.
Advantages:

One could argue that SH-SY5Y cells are pseudo-neuronal cells, which is why they are commonly used to study neuronal diseases such as Parkinson's disease and other neurodegenerative diseases. [3, 4]

Disadvantages:

The cell line is not personalized, i.e., it's impossible to get cells from depressed and non-depressed patients, thus it's tricky to find biomarkers for depression, if you cannot compare a "healthy" with an "unhealthy" protein expression.

Question
Do you know any readily available cell line that could be used to find differentially expressed proteins in depressed subjects that is close to neuronal cells? Neuronal cells from the cerebrospinal fluid (CSF-contacting neurons) would probably be the best one could get from living subjects, but the setup of an approveable study and the recruitment of subjects that voluntarily accept a lumbar puncture is a bit tricky to say the least.

tl;dr there is no 'depression' cell line. Cell lines would be suited for studying pharmacology, cellular or molecular mechanisms, none of which are depression. Depression also has no good correlates on the molecular level which can be used as a proxy by virtue of using in vitro cell lines. For instance, you can use cell lines to study amyloid protein biosynthesis or clearance to better understand the proteostasis that occurs in neurodegenerative disease, which are thought to arise through aberrant proteostasis. But depression itself is poorly defined and is less characteristic than that on a molecular level, and I certainly don't know of any cell lines that can be used to study "differential expression of genes in depressed folks". You'd be better prepared by taking brain biopsies and even that would be very incomplete of a picture to begin a study.

Hi Sam. Really interesting idea, you certainly pose the question diligently. Do not be discouraged from thinking about such things following a read through my response. But... I think many first impressions here would include critical thoughts about how you formulate and think about the experiment.
Thus, I would challenge your question. First, depression is a syndrome, and is not very clearly defined, certainly not well at the molecular level. If you want to reduce depression to a mechanism study-able in cell lines, you first need to justifiably operationalize it somehow. This I think is very problematic already.
Additionally, depression does not occur at all in cell lines, or even between neurons in synapses, but rather (loosely speaking...) in vast, interconnected circuits, as its readout - symptomatology - are observable only as the output of entire nervous systems, in organisms. Depression is not caused by faulty enzymes as results of single mutations; they are very highly multi-faceted. As such, cell lines cannot be used as proxies. What cell lines can be used for is to study specific molecular interactions, say with how pharamaceuticals are metabolized, or which compounds may impact proliferation. However, here I quote a few things from one of your sources that jump out even during a quick skim read:

On the basis of the neurotrophic hypothesis of antidepressant’s
action, effects of antidepressant drugs on proliferation may serve as
tentative individual markers for treatment efficacy
Peripheral proliferation is unsuitable as surrogate marker for
antidepressant response
Although the in vitro treatment of patient-derived LCLs with fluoxetine presents high inter-individual variability regarding the LCL proliferation behavior, this phenomenon has—according to our data—no association with the patient’s clinical outcome.

The paper merely screened for differential expression in genes in white blood cells following fluoxetine exposure, and found only two candidate genes, SULT4A1 and WNT2B, which they have some reason to believe may be involved in drug metabolism, which were correlated to a donor clinical response and remission, respectively. This is a paper which suggests potential candidates for further study, but does not at all implicate or demonstrate any connection at all between depression or the efficacy of the drug, or the genes involved. It is meager in lieu of your question! The key here is that they compare cells before and after fluoxetine exposure, not that they are comparing depressed or non-depressed neural tissue. They tried to use remission to correlate things, but it just didn't work well. Which I think is expected!

Thank you for this very detailed answer. Reading your take on this gives the impression that you don't think that it's even possible to study depression in cell lines. This may be true if you want to correlate your findings with a clinical outcome but it's certainly not true if you're interested on the cellular level only. It's clear that depression is a very complex syndrome and I think that it's also safe to say that many factors play an important role here, but nevertheless there are multiple studies showing that subjects with a diagnosed MDD do show differentially expressed proteins. (1)

A polymorphism within the promoter region of the serotonin transporter gene (SERTPR) was thought to be a risk factor for the development of depression (Mandelli et al 2007). Significant associations have been described between single nucleotide polymorphisms in the FKBP5 gene and antidepressant response and recurrence of depressive episodes (Binder et al 2004). A polymorphism in the GNB3 gene was also found significantly more often in depressed than in healthy controls (Zill et al 2000). This shows that it's safe to say that there are proteomic differences in depressed subjects. (2)

We want to take this further by determining potential biomarkers for depression by bottom-up LC-MS analysis from neuronal cells to subsequently identify possible long-term risk factors, for example, long-term use of specific drugs. For this purpose, cells are needed that can be incubated with the drugs. (3)

As a counter-example to your opening sentence, see Lithium-responsive genes and gene networks in bipolar disorder patient-derived lymphoblastoid cell lines, published in Nature in 2016 (Lithium has been used as a very effective drug in the management of manic depression since the 1960s)

@Sam You can look for biomarkers in a cell line but how do you know they have anything to do with depression? Your cells won't be depressed. You can certainly study a gene of interest in a cell line, but still that cell line won't be depressed. The cell type you use probably doesn't matter much - HEK cells, primary neurons, whatever you want, they won't be depressed either, they will just express your allele of interest.

@BryanKrause That's absolutely right. That's why the LCL cells are so interesting. With them it is possible to get cells from a depressed and a non-depressed subject and to compare the protein expression in both (actually you'd obviously need more subjects in each group). This would then allow to correlate the differential protein expression with the clinical manifestation of a depression. But as depression is something psychological, I guess that neuronal cells are way more interesting than lymphocytes. (1)

@BryanKrause We have to remember that each cell type expresses a different set of proteins. I think that it's clear that neuronal cells are way more likely to show differences in their protein expression, that are in fact related to the manifestation of a depression, than those of lymphocytes do. That's why I was interested in different cell lines that may combine the neuronal aspect of SH-SY5Y cells and the easiness of individual production of LCL cells. (2)

@Sam Your question asked about cell lines, not about which cells to isolate from patients (and controls).

@BryanKrause Yeah, I wasn't clear enough in my explanation, I'll edit the question, but in short my problem is that I have to do both, search for biomarkers by bottom-up LC-MS (untargeted) and check whether or not these proteins are differentially expressed in normal, i.e., "healthy" cells when the cells are incubated over a long period of time with pharmaceuticals of interest. At least for the latter project, I'd need a cell culture to be able to incubate the cells with these substances.

Hi Sam - I see your points, cell lines can be relevant for depression research in some way. Interesting ways too. However, I side with @BryanKrause, you can only study correlates of depression with cell lines, not depression itself, and I am under the impression that you suggest you are using 'depression cell lines' as opposed to just looking at gene expression in unrelated cells following pharmacological treatments. Certainly there are some reasons why LCLs, but they are only convenience and approximation; as long as you are fully aware of (and open about) that, the science is fair game!

@sam A big problem I see in this is that the cells will change with culture, so correlates outside of the body will be different to those found inside the body, even if you could isolate cells from a depressed person, there is no guarantee that those cells will maintain the phenotype or that they are actually the "depressed" part. If you want truly neuronal cells you might try iPSC neurons, my understanding is that these are as close to true neurons as you can get without getting actual brain tissue.


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Emotion as a conscious prediction error signal?
At first glance it seems many if not all emotions can be explained as prediction errors: hunger the observed level of energy stores is below expectation, anger the observed level of “respect” was below expectation, fear the observed level of personal safety was below expectation, disdain the observed level of respect afforded another person was above expectation, and so on. In addition, this explains the “motivational” component of emotion, namely to alter beliefs or execute actions to eliminate the prediction error in line with Friston’s free energy principle. Eg in the case of hunger, to eat, in the case of fear fight or flight, in the case of anger either dispute the offense to obtain a retraction or punish the offender, and so on. So I’ve found it surprising that it appears difficult to find research related to explaining emotions as “active inference”. The closest I’ve found are Emotions as computational signals of goal error  and Emotion and the predictive mind: Emotions as (almost) drives.
Is this an active area of research? Any pointers are appreciated.

Please edit the question to limit it to a specific problem with enough detail to identify an adequate answer.

Definition of emotion used is somewhat vague: is hunger an emotion? It has to do with real physical needs, like physical pain, whereas the other examples have to do with mental activity/relationships. Also, what about love, hate, curiosity, satisfaction, and many other emotions?

Well that’s the essence of the question.  Can some (or all) emotions be defined as conscious prediction error signals, where the predictions are the brain’s expectations about what it will observe. The expectations include priors like physical needs (which yield feelings such as hunger, thirst, oxygen hunger, salt hunger, pain, etc when they are violated) as well as learned beliefs about the causes of what it observes, which can be very high level explanations. The list is too long for this comment, but for example, embarrassment, my observed level of “competence” is below expectation.

It seems someone could define things this way for at least some emotions, but then....what's next? Why is that useful from a biology standpoint? There are predictive coding models for the brain but these are specified in terms of how the prediction errors propagate information within the brain.

Well, the obvious answer is that it could lead to a principled explanation of emotions in terms of free energy minimization, and if correct eventually to identification of the prediction error units and neuromodulators responsible.

@ChristopherOliver That doesn't make a whole lot of sense to me, but seems to be stretching Friston's ideas further than even Friston would.