text:
Which mold species are these?
A friend of mine in Los Angeles recently put a petri dish with some mold-friendly substrate under a vent for two days, and a whole rainbow of mold species grew on it.

What are the different mold species present on this dish? Are any of them dangerous to human health?

you need to give a lot more information and if possible take pictures with 50x magnification of each type in your sample.

I'm not sure the OP can give "a lot more information" except maybe what the "mold-friendly substrate" was, but I think an answer linking to a resource for common household mold species would be a wonderful and helpful answer. +1 for home experiments!

@trondhansen - I am happy to give any other information that I have and that might be relevant, if you tell me what you are looking for. I did not take the picture, and my friend does not have a microscope, so I cannot easily get you 50-times-higher-resolution pictures. I imagine some "best-guess" answer might have to do if there are various mold species with similar phenotypes.

The wet pink ones are likely to be either a yeast or something like Serratia marcescens. We would need microscope pictures of stained samples from the others to attempt ID. Filamentous fungus is about as close as you can get otherwise. Huge range to choose from...


text:
Who discovered that phage phiX174 has a single-stranded genome?
I have recently read that Erwin Chargaff has discovered that the genome of phage phiX174 (ϕχ174) is single-stranded. However I could not find the paper reporting this discovery.
Is there a link to the paper reporting this finding?
Thank you.

can you give the citation, quote, or other paper trail for us to follow? Finding old papers can be difficult, and it isn't clear even whether Chargaff made that finding or merely if his work informed the discovery. I cannot find any meaningful association of him to the phage, though I also can't find an obvious paper or reference on pubmed that points to the discovery. Knowing what your starting point is would help.

I think @MaximilianPress was close. Chargaff certainly did early work on DNA structure, but he was not the one to identify that the genome of φX174 was circular.  As far as I can tell, the actual peer reviewed publication was a pair of papers in Journal of Molecular Biology, written by R.L. Sinsheimer in 1959. I can access these articles through my institution, but I suspect that they are paywalled.
These two articles are in volume 1, issue 1 of the journal on pages 37-42 and 43-53 respectively. I've reproduced the citations with abstracts below. The first indicates some oddities in the DNA of the bacteriophage and the second confirms these oddities and provides the explanation (emphasis is mine to highlight the salient point).

Robert L. Sinsheimer,
Purification and properties of bacteriophage φX174,
Journal of Molecular Biology,
Volume 1, Issue 1,
1959,
Pages 37-IN5,
ISSN 0022-2836,
https://doi.org/10.1016/S0022-2836(59)80005-X.
(https://www.sciencedirect.com/science/article/pii/S002228365980005X)
Abstract: Procedures are described for the isolation in pure form of the bacterial virus φX174. This virus is shown to have a particle weight of 6·2 × 106 and to contain 25% by weight of DNA. At neutral pH, the purified virus forms a discrete aggregate, probably a tetra-mer. This aggregate dissociates either upon dilution or at alkaline pH. Lysates produced by φX174 contain a second particle, antigenically related to the virus, but not infective and of lower sedimentation rate and lesser DNA content. The DNA of φX174 appears to have an unusual structure in that it reacts with formaldehyde (even before extraction from the virus) and in that the atomic efficiency of inactivation of the virus by decay of incorporated phosphorus-32, calculated by combining our data and that of Tessman, is 1·0.

Robert L. Sinsheimer,
A single-stranded deoxyribonucleic acid from bacteriophage φX174,
Journal of Molecular Biology,
Volume 1, Issue 1,
1959,
Pages 43-IN6,
ISSN 0022-2836,
https://doi.org/10.1016/S0022-2836(59)80006-1.
(https://www.sciencedirect.com/science/article/pii/S0022283659800061)
Abstract: The deoxyribonucleic acid (DNA) of bacteriophage φX174 can be extracted by phenolic denaturation of the virus protein. The DNA thus obtained has a molecular weight of 1·7 × 106, indicating that there is one molecule per virus particle. This φX DNA does not have a complementary nucleotide composition. The ultraviolet absorption of this DNA is strongly dependent upon temperature in the range 20° to 60°C, and upon NaCl concentration in the range 10−3 to 10° M. This DNA reacts with formaldehyde at 37°C and is precipitated by plumbous ions. This evidence is interpreted to mean that the purine and pyrimidine rings are not involved in a tightly hydrogen-bonded complementary structure. Light scattering studies indicate that this DNA is highly flexible and that its configuration is strongly dependent upon the ionic strength of the solution. Upon treatment with pancreatic deoxyribonuclease, the weight-average molecular weight decreases in accordance with the function expected for a single-stranded molecule. It is concluded that the DNA of bacteriophage φX174 is single-stranded.

well done! this definitely the right answer. Somehow I got the author/date right but clicked on the wrong article?

@MaximilianPress Thanks. Took a bit of digging to find them because they apparently aren't indexed on Pubmed - it only comes up with the symposium you found, which is odd as J Mol Biol isn't exactly an unknown journal with no publishing history.

yeah, that must be what happened to me! And then when I saw author-date matching in the reviews I assumed it was the same. I also think that the "cited by" PubMed feature got confused for similar reasons, and linked me out to the papers citing the right (JMB) paper by accident! What a tangled web we weave.

Thank you. Sorry but I did not have much quotes to report.

Update
please see @bob1's answer below.
Original answer
I suspect that this is the paper you are interested in but I can't find even an abstract of it due to the obscurity/loss of the journal.
In a different review paper, this 1959 Sinsheimer paper is described as such:

The first DNA molecule purified to homogeneity was the genome of bacteriophage ϕX174, reported by Sinsheimer in 1959 (12). Equilibrium buoyant density centrifugation of the ϕX virion yielded pure preparations from which the DNA could be easily isolated by phenol extraction. ϕX DNA turned out to be a single-stranded circular molecule that was estimated to be ∼5000 nt in length.

It’s not a journal but a symposium volume. Most big university libraries would have it secreted in the bowels of their archives. I’ll see if ours has it, just for fun.

@David thank you! that would be wonderful.

I've found it in Google Books at https://books.google.co.uk/books?id=6vm1eM7-zJYC&pg=PA1&source=gbs_toc_r&cad=2#v=onepage&q&f=false  If anyone has difficulty with this link I've taken screenshots and can mount a pdf on one of my websites. Chargaff wasn't present at that symposium, according to the list of participants at the end. I think the poster has probably got hold of the wrong end of the stick. Chargaff was interested in this because its base composition violated the so-called Chargaff Rule. However we now know that this reflects the base-pairing in dsDNA. I can post an answer if useful.

source: Stack Exchange

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

sort: votes


text:
Bacterium fixed nitrogen transfer mechanism
As it is well known, Rhizobium and other bacteria have the capacity to fix Nitrogen and thus perform a crucial role in the natrual nitrogen cycle.
I am trying to find a reliable source on how the origin of the nitrogen that the bacteria fix, for example inside the nodules.
Most sources simply explain how nitrogenase converts N2 (in gaseous form) to NH3. But I have not found a source explaining the origins of N2 in the soil environment.
I don't see how the N2 could be diffused in the soil from the atmosphere (it would need impractical large time for such diffusion, and morever that would negate the soil aeration in general)
An agronomist pinpointed the N2 relased in the soil during decomposition, but that N2 would not be a net general fixation (from the atmosphere to the soil as a biological useful nitrogen)
The solubility of N2 into water could explain some net transfer on atmosphereic N2 into the soil, but it is relatively small amount (20mg/L)
So my question is where does the N2 in a plants root environment comes from?
Do the plants transfer N2 to the roots dissolved in the phloem?
I would appreciate and insight and/or any sources.

related to your question,your bacteria replaces the ammonia step in this cycle  https://en.wikipedia.org/wiki/Nitrogen_cycle

What sort of structure do you think soil has? Is it a solid mass like a lump of plastic or metal, or does it have pores/holes/granular structure?

air contains 78% nitrogen(N2),bacteria converts this to nitrate that the plants can use.https://en.wikipedia.org/wiki/Soil_gas

Thanx for all the comments. The answer to my question seems obvious now. My assumption on the diffusion time is not correct, due to the pore structure of the soil.

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


text:
What Causes the White Layer on These Leaves?
I recently noticed that a Malabar Java plum (Jamun) tree in our garden has most of its leaves covered in a strange white layer, like in the picture I've attached.

I'm curious to understand why this is occurring and whether it could potentially lead to allergies such as allergic rhinitis. What's causing this, and what can be done about it? Any help understanding this would be great. Thanks!

Please read about powdery mildew to see if that fits what's going on with your plum. Without handling the leaf, I can't be sure, but it's the most common cause of white, grey or brown spots on plants.

You also have scale insects on there (along the mid-rib are the most obvious adults, but lots of life stages all over in patches) - these are sucking insects, often covered in white waxy or powdery substance.

@bob1 - Wow, I need new glasses; I completely missed that. Scale insects also exude a sweet substance that promotes fungal growth, but I think the primary problem there is scale.

@anongoodnurse Yep, scale looks like the problem - you can even see where a couple of adults have come off mid-leaf, leaving the rings behind. Feel free to write it up.

Thanks, but I missed it, you caught it. :)

The major problem your plant has here is a very heavy scale insect (Coccomorpha) infestation. These are sucking insects that bite into the veins of plants and suck the sap. They are related to aphids, and feed in a similar manner. The females attach themselves to the plant and are covered by a shield and/or a waxy layer for protection from the environment and from predators. Like aphids, when feeding they exude some sap residue from their gut and this can result in growth of fungal species on the plant. The fungal species are often black sooty molds, but I would hazard a guess that not always.
You can see adults clustered along the main mid-rib of the leaf and smaller instars clustered along the lines of leaf veins. The adults look like ovals with a darker line running down the middle. The instars are particularly obvious about mid-leaf, near the two ovals outlined in white, where you can see 3 approximately regularly spaced veins with lines of them feeding. The two ovals outlined are where adults have fallen off and left behind some of the waxy coating they coat themselves with.
I don't know which of the many many species of scale insect within the grouping you have here, but one common and well known grouping within the scale insects is the mealybug (not what you have here, mealybugs are all unarmoured, yours is armoured). You should be able to treat your plant with a product that is designed to kill mealybugs. These products usually contain an oil delivery system to counteract the waxy water repellent layer the scale insects coat themselves in, and an insecticide to kill the insects.
The white residue you are seeing on you plant is probably not mold and is the waxy substance you can see on the insects. I can't find any reports in the scientific literature of allergy to scale insects, so these are unlikely to be causing a problem. Molds from the secreted sap might cause a problem as fungi from many types are known to cause allergies.


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How would an animal with legs longer than its torso perform?
The horror game Poppy Playtime features a monster called Catnap. Simply put, the creature has legs far longer than its torso, like a giraffe but minus the long neck (its based on a cat).
The game shows Catnap as being quite agile. However, looking at him I wonder if something with that sort of bodyplan could really move all that well. I know giraffes can't run very fast, though given that Catnap doesn't have an overly long neck, I don't think that would be a great real-world analogue.
The game mainly shows him walking around on all fours, even though he is shown standing up straight in one brief scene. In one of the few scenes where you can see him running, he seems to be using a galloping motion. He's also shown being able to climb rather adeptly, and even fit into rather small spaces.
Keep in mind, monsters like Catnap in the game aren't entirely biological. The game doesn't make it clear how much of the is organic and how much is mechanical. Some of the monsters clearly has impossible body plans, like giant heads connected to the body by extremely thin necks, but somehow still able to swallow humans whole. Seriously. Also, the monsters are supposed to be humans transplanted into giant toys. Given this, its highly doubtful that Catnap's overly long limbs are entirely organic, though it does appear to have an analogue for a skeleton in it.
Its obvious that the devs are more designing the monsters to look creepy than anything else. Catnap though really makes me scratch my head; would he really be as agile as he's shown in the game with such cartoonishly long limbs? At least with similar creature the arms are clearly more tentacle-like, but his limbs aren't shown as such. He does actually appear to have elbow and knee joints. His limbs also stay staight most of the time, implying that he does have actual bones or some similar in his limbs. Point I'm making is regardless of how much of him is actually organic, it would probably be safe to treat this question as if he really was.
So, how would an animal perform with a bodyplan like that? Would a giraffe be more agile if it didn't have that overly long neck? That's basically what catnap is (minus the fact that he also doesn't have hooves, being a cat and all). Even without a neck though he's still rather towering. Its hard to get a concept of scale, but based on another toy whose measurement is given, he probably stands around 20 feet at the head when standing upright, just to give you an idea of his scale. Even on all fours, he towers over the player. His back almost touches the ceiling in some interior areas, though he's still shown walking on all fours even in open areas. The game even starts with him carrying the player in his mouth, rather than his arms. Perhaps his overly long limbs just make it too hard for him to stay standing upright? He never does walk standing upright.

You might get a better reception for hypothetical questions if you asked this on our [worldbuilding.se] site. It's for helping writers/artists/game-designers create their world - seems the sort of thing you have here that you need help with. Be sure to take their tour, read their help centre etc. before posting. ... Ahh, seems you're a member already.

Your actual question about mechanics is a good one for Biology SE. However, folks here don't like a lot of background or "what got me thinking about this is..." material, thus the down votes and currently 2 close votes. You can improve the reception of your question here by removing most of that stuff, which would be perfectly fine in a different SE site like Worldbuilding. Going from one site to another is like going from one country to another; new customs, traditions, and behaviors that offend the locals. :-)

Short answer: creatures with such morphology and features (except for hunting humans and climbing very well) already exist, and yes, it's the Giraffes.
You see:

Giraffes are actually quite fast, they can run as fast as 35 mph (which equals about 56 km/h), which is about the speed of zebras, which also reach speeds up to 35 mph. Of course there are substantially faster animals, wild horses for example clock in at up to 54 mph, however this is little surprising, as wild horses are so well adapted to running humans domesticated them for transportation.

Funnily enough, short-necked giraffes even exist (and still have long legs despite having a short neck, as you can see here), and given that normal giraffes are already quite fast, it seems reasonable to assume that the neck does not hinder the giraffes while running and the short-necked ones are about as fast as their long-necked counterparts.

Interestingly, giraffes can even climb hills to some extent, even tough they're not good climbers.

So actually, (short-necked) Giraffes probably already possess most of the features of Catnap, except for the good climbing skills and hunting people.

To be clear, catnap isn't 'mine'. Its a character in a horror game franchise. I didn't make him and I don't own him.

Of course, I didn't consider that this phrasing could be understood that way. I edited my answer accordingly.

There's also the giraffe relative, Okapi. Their legs aren't so long, but about the same as the torso.

Those images look photoshopped to me. Also, when I was little, I saw a tv program showing humans competing against animals (seriously). One of the segments had a human race a giraffe. The human beat the giraffe by a mile. He was beat by a zebra (obviously). Given that humans could only run up to 20~25 mph (this was a full decade before Usain Bolt just so we know), that giraffe clearly wasn't running at 35 mph. It was probably running at about half the speed of the human. I call bs on all this post.

Oh, and looking it up, I found that very clip! If a giraffe can run at over 30 mph, then how do you explain this: https://www.youtube.com/watch?v=Y5QwYJoLUfE

The giraffe has not much of a reason to run at top speed. If it was chased by a predatory animal, it would probably go much faster.

See also maned wolves.

Kinda are similar to the Catnap described and is actually predatory. Didn't know such a thing exists, cool.


text:
What distinguishes a colony from multicellular organism?
I would like to inquire what made biologists conclude that colonies such as the famous Volvox is an organism constituted by independent cells as opposed to a multicellular being.
I read that it has something to do with "degree of independence". However, what exactly are the parameters of a colony and the so-called "degree of independence" between cells and how is it different from multicellular beings despite possessing notable differentiation such as the latter?


text:
How to resolve the contradiction between the time required for the fixation of neutral mutations and the time available according to the fossil record
More than thirty thousand synonymous single-nucleotide-substitutions have occurred in protein-coding genes in the human genome since the human lineage diverged from the chimp lineage ~7 million years ago (Bakewell et al, 2007, p.7490). Since, by definition, synonymous substitutions don’t change the amino acid, these substitutions must be selectively neutral mutations. For neutral mutations, the rate of evolution is simply equal to the point mutation rate U, “irrespective of selection at other linked loci, changes in population size, or almost any other conceivable complication” (Lanfear et al, 2014, p.36). This is problematic because U is approximately 10 to the -8 power for humans, which means that it took about a hundred million generations for each of these substitutions to become fixed in the human genome. Since each generation is more than ten years, a hundred million generations is more than a billion years, which is much longer than the ~7 million years since the human lineage diverged from the chimp lineage. How is this to be reconciled?
Bakewell, M. et al, “More genes underwent positive selection in chimpanzee evolution than in human evolution.” Proceedings of the National Academy of Sciences USA, vol 104, pages 7489–7494, 2007
Lanfear, R. et al, “Population size and the rate of evolution.”
Trends in Ecology and Evolution, vol 29, pages 33–40, 2014

Comments have been moved to chat; please do not continue the discussion here. Before posting a comment below this one, please review the purposes of comments. Comments that do not request clarification or suggest improvements usually belong as an answer, on [meta], or in [chat]. Comments continuing discussion may be removed.

The parameters cited by OP are not in conflict.
The misconception relates to a disconnect between what the theory predicts and the type of observation we have available. This leads to an unnecessary assumption that the observed substitutions have become fixed, as well as a failure to account for standing variation at the time of divergence.
Lanfear et al. correctly states that "the rate of evolution at a site is $KU × 1/K = U$". To find the amount of (this type of) evolution between two populations over time, one would calculate $UTG$, where $T$ is the time in number of generations, and $G$ is the genome size. However, this is a calculation of neutral substitutions that have become fixed across two diverged populations, and is really most appropriate when $T$ is much greater than the expected time to fixation ($T>2N$, where $N$ is the effective population size).
To perfectly test the theory, one would need to census all substitutions in both populations in order to differentiate between substitutions that have become fixed and those that are polymorphic.
The observation we have here, however, is essentially a single individual from each population.
When one compares two individual genomes that have been separated, the SNPs observed between them will include:

sites with a SNP that originated since divergence and has become fixed (what the theory is talking about).
sites that are polymorphic, with SNPs that originated since divergence.
sites polymorphic in the original population, where a SNP became fixed in one lineage and lost in the other.
sites polymorphic in the original population that are still polymorphic.

SNPs originating since divergence that have become fixed
Even just applying the above calculation, considering only fixed SNPs (or those that will become fixed) provides the same order of magnitude as the observation of 30,000 substitutions in humans since divergence with chimps.
Bakewell et al. surveyed 5,215,415 synonymous sites, so we'll use that as $G$. Assuming 20 years per generation and 7 million years of divergence $7×10^6/20= 3.5×10^5$ generations:
$UTG = 10^{-8} × (3.5×10^5) × 5,215,415 = 18,253$ substitutions (though we don't know which of the 30,000 observed substitutions these are).
Sites that are polymorphic with SNPs that originated since divergence.
New mutations are entering the population all the time, with every individual.
Imagine this measurement was taken one generation after divergence, and where the ancestral population was all exactly identical.
The human genome is $3×10^9$ nucleotides, so $10^{-8} × 1 × (3×10^9) = 30$ substitutions. Humans and chimps are diploid, so an individual would have ~$30$ mutations on each set of $3×10^9$ nucleotides, or ~$60$ mutations compared to our hypothetical source population.
As Lanfear pointed out, each one of these substitutions is unlikely to eventually become fixed (they have probability $1/K$), but we're taking the measurement now, so we observe them. Many of them are likely to spread in the population at least a little bit, even if they eventually disappear.
We also know from Bakewell that both the observed human and chimp genomes contain sites that are not only polymorphic in the population, but polymorphic in the sequenced individuals.
Sites polymorphic in the original population, where a SNP became fixed in one population and lost in the other
Overlooked by OP is the fact that the most recent common ancestor of humans and chimps (which was a population, not an individual) was not full of identical individuals, but contained the standing genetic variation one sees in any species. This allows for the fixation of alternative alleles between the daughter populations, even in the absence of new substitutions.
We know in fact this happened. The gorilla genome showed us that ~30% of the gorilla genome is more closely related to human or chimp than the later are to each other. This is due to a phenomenon called incomplete lineage sorting, which occurs when two speciation events occur over a short span of time (again $T<2N$; the gorilla/human/chimp split is thought to have occurred ~10 million years ago). It is caused by variation in the ancestral population that gets differentially sorted into the daughter populations.
Sites polymorphic in the original population that are still polymorphic
It's possible there are still sites that retain polymorphism within the daughter populations that was present in the ancestral population. A wide survey of human and chimp genomes could find these alleles.
(This may be rare between humans and chimps. Estimates of the human effective population size are generally $<10,000$, so $2N=20,000$. Our estimate of ~350,000 generations probably means few sites in the genome have failed coalesce in that time.)


text:
Is there any plausible mechanism through which magnetic storms can directly affect the health of people on the ground?
https://ui.adsabs.harvard.edu/abs/2014cosp...40E1114G/abstract#%3A~%3Atext%3DIt%20was%20shown%20statistically%20that%2Ccomparison%20with%20quiet%20geomagnetic%20conditions
This study says that on average, rates of heart attack and brain stroke increased by an average factor of two(!) during geomagnetic disturbances, due to Earth's magnetism.

It was shown statistically that during geomagnetic disturbances the frequency of myocardial infarction and brain stroke cases increased on the average by a factor of two in comparison with quiet geomagnetic conditions.

They offer this mechanism for this observation, and the mechanism involves magnetism:

Our investigations show the red blood cells are very sensitive to electromagnetic forces. Most probable that geomagnetic fluctuations (frequency) acting on blood, brain, adrenals involves the adaptation system. This leads to increasing a level of catecholamines in blood responsible for activation of the clotting system, rise in aggregation and spasm in the afferent vessels of the microcirculatory network. In persons suffering from CHD, the foreground problem is the reversibility of these pathological processes.

On the other hand, USGS, a government domain website, asserts that Earth's magnetism does not affect health. The subject is on the topic of magnetic storms.
https://www.usgs.gov/faqs/does-earths-magnetic-field-affect-human-health

"Does the Earth's magnetic field affect human health? The Earth's
magnetic field does not directly affect human health. Humans evolved
to live on this planet.
High altitude pilots and astronauts can experience higher levels of
radiation during magnetic storms, but the hazard is due to the
radiation, not the magnetic field itself. Geomagnetism can also impact
the electrically based technology that we rely on, but it does not
impact people themselves.
Learn more: USGS Geomagnetism Program"

It says that geomagnetism can affect electrically based technology, but it does not affect people themselves.
Health effects are not listed in their page titled "What are the hazards of magnetic storms?"
https://www.usgs.gov/faqs/what-are-hazards-magnetic-storms
Is it plausible that Earth's magnetic storms causes the rate of heart attacks to double? Is the mechanism suggested by https://ui.adsabs.harvard.edu/abs/2014cosp...40E1114G/abstract#%3A~%3Atext%3DIt%20was%20shown%20statistically%20that%2Ccomparison%20with%20quiet%20geomagnetic%20conditions in any way plausible?

What is the base rate?

The second author has been publishing on these sorts of things for a while Breus T, unfortunately the papers are largely not available or in Russian, so hard to access for me. From abstracts, data seem sketchy at best.

Cross-posted to skeptics here. Please don't clutter the network with duplicates. I suggest confirm the truth of the statement on skeptics, then come here to ask the mechanism by which it happens - once it's been confirmed to be real. Best to delete this here now.


text:
What is the genus of this fungus?

I'm struggling to identify this fungus. Is it Aspergillus sp. or Penicillium sp.?

Welcome to the Biology Stack Exchange. You would need to provide much clearer photos and some photographs down a microscope of the structures seen, particularly the sporangia for ID.


text:
Bile storage and secretion
I know that the liver has left and right hepatic ducts that unite forming the common hepatic duct that then unites with the cystic duct of the gall bladder forming the common bile duct which unites with the pancreatic duct forming the ampulla of Vater that opens in the duodenum.
I also know that the liver secretes its bile which is stored in the gall bladder.
My question is how come the liver secretes its bile via the hepatic ducts and stores it in the gall bladder at the same time?

Because there are two main functions of bile in the body, and in this way, both can be achieved. Googling functions of biliary system should give you the answer,. If you edit to provide a quote and a link to the source(s) you found, and still don't understand, please explain what you don't understand, receive a more complete answer. Thanks.


text:
Does inoculum volume affect spread plate outcome?
Background
The spread plate method is a technique used in microbiology to enumerate and isolate bacteria or other microorganisms present in a sample. It involves evenly spreading a liquid sample containing microorganisms onto the surface of a solid agar medium in a Petri dish.
Inoculum Density: Inoculum density refers to the concentration or density of microorganisms (bacteria, fungi, etc.) present in the sample that is being plated onto the agar surface. It is typically measured as the number of colony-forming units (CFUs) per unit volume of the sample.
Inoculum Volume: Inoculum volume refers to the volume of the sample that is spread or plated onto the surface of the agar medium during the spread plate method. It is typically measured in microliters (µL) or milliliters (mL).
Question
Does inoculum volume affect spread plate outcome?
If I pipetted 0.1ml 0.5 McFarland of bacteria into the agar plate before spreading, would it differ from pipetting 0.5ml of the same density?

Theoretically, for CFU determination the volume of the inoculum shouldn't matter as long as you plate a dilution series and find the plate with a countable number of colonies. It also shouldn't matter if you use the spread-plate method or the pour plate method or something else (like drop plates or spiral plates). But all of that assumes ideal conditions where you are able to get a perfect and uniform spread over the whole plate every time. In the real world there are practical limitations on the range of usable inoculum volumes when spreading liquid onto agar plates.
When making spread plates it's best to maintain continuous spreading motion until all (or almost all) of the liquid from the inoculum has been absorbed by the agar medium. If you don't, you can end up with unevenly distributed colonies, which can make otherwise countable dilutions difficult or impossible to count accurately. If the volume is too large, it may take an impractical amount of time for the liquid to absorb. On the flip side, the volume still needs to be large enough to cover the whole plate evenly. If it's too small (say 10µL) you may have trouble spreading it evenly across the entire surface.
There's also some potential for variation in consistency when dispensing different volumes. Most micropipettes are slightly more accurate at the high end of their range than the low end. So, IF you were using the same p1000 pipette, your replicates could be slightly more consistent with larger volumes than small ones. But, properly calibrated, the dispensed volume should vary by less than 10% even at the lowest setting of most pipettes, and if this were a concern, it could be remedied by switching to a more appropriately sized pipette.
Lastly, if you are trying to make a bacterial lawn, for something like a Kirby-Bauer test, changing the inoculum volume can affect the outcome of the test. McFarland standards aren't always perfectly calibrated for every organism, but overloading the plate with 5x the standard inoculum certainly has the potential of skewing the test results. The methods for these tests are standardized to improve repeatability between labs. Essentially, a denser inoculum will reduce the size of the inhibition zone, while a reduced inoculum will increase the inhibition zone. Since the inhibition zone determines whether a particular isolate is classified as resistant or susceptible to a given drug, inoculation protocols are standardized to make sure patients receive the appropriate care.
Source: EUCAST Disk Diffusion Method for Antimicrobial Susceptibility Testing  - Version 9.0 (January 2021)

Thanks for the great answer. I'm trying to do the last: bacterial lawn for Kirby-Bauer test. Could you reconsider that it won't affect kirby-bauer test? I'm saying this by gut feeling. It must have to do something with the McFarland standard concentration being fixed at 0.5 for the kirby bauer.

@FreezingSoul I see. It's worth mentioning that AST protocols typically call for inoculating lawns with sterile cotton swab dipped in the bacterial suspension, rather than spreading a specific volume on a plate. I'm sure there's some variation in the actual volume transferred from the swab to the plate, but probably not on the order of the 5-fold difference specified in your question. See updates to my answer for more specifics on how inoculum density can impact the observed inhibition zones (I confess that I haven't evaluated those statements personally).

I tried to keep the question as abstract as possible so more people would benefit from it. But, my problem is that I think my agar soaks the 0.1ml so fast that I don't have time to spread it. Maybe because I dry them by incubating them for 24 hours after pouring, which I don't plan to give up, because it's primarily a sterility check. Is there any problem if I add my 0.1 inoculums to some 0.5ml saline before spreading and spread the whole 0.6ml, since i can't withdraw more inoculum volume?

i think nevermind, the problem may be that i waited to too long for the spreader to cool, it says in a textbook that you got 15 seconds after pipetting the inoculum to spread otherwise the water will be soaked.

To add to the answer by @MikeyC . The spread plate technique for calculation of colony forming units has volume plated as a variable:
CFU/ml = (number of colonies x dilution factor) / volume of culture plated
This means that you account for the volume in the calculation performed after doing your counting.
Of course, as MikeyC mentioned, the volume matters to some extent as if you add too much of too high a density suspension, then you will have an uncountable plate. To compensate for this, what is usually done is a dilution series, where you choose which dilution is used for the calculation This also provides a secondary check on your count, as a plate below (lower dilution) the countable dilution should be TMTC and the one above should have approximately 1/(dilution factor) fewer colonies. For example, in a 10-fold dilution factor series, your countable plate might have 50 colonies, the one below should have ~500 (TMTC) and the one above should have about 5.
You should also note that the dilution factor is also compensated for in the equation.


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Reading genotype aloud
For this genotype, P2ry12$^{+/+}$ I read it "P2ry12 wild type." For animals with the GFP being expressed under one Cx3cr1 promoter, Cx3cr1$^{+/GFP}$, how do I read that aloud?
What about for: Cnr1$^{flox/flox}$, Cnr1$^{flox/+}$, Cx3cr1$^{CreER/CreER}$, and Cx3cr1$^{CreER/+}$ mice?
I'll give my attempts below and take suggestions:
Cx3cr1$^{+/GFP}$ "Cx3cr1 wild type with heterozygous GFP expression" or "Hetero GFP expression" or "GFP" as in for P2ry12$^{+/+}$, Cx3cr1$^{+/GFP}$ animals it would read "P2ry12 wild type with GFP" to reduce redundancy if it is clear to the listener.
Cnr1$^{flox/flox}$  "with Floxed Cnr1"
Cnr1$^{flox/+}$ "with hetero floxed Cnr1" Would the words incomplete or partial ever be appropriate here? As in "a partially floxed Cnr1?"
Cx3cr1$^{CreER/CreER}$ "homo CreER under the Cx3cr1CreER promoter" or just "the CreER animals"
Cx3cr1$^{CreER/+}$ "the hetero CreER animals"
and for CreER is it correct to say that is a chimeric protein of Cre and mutated hormone-binding domain of the estrogen receptor?
Thank you in advance!
Edit:
I do see that one of them is clarified by the authors: P2Y12-deficient (P2ry12$^{–/–)}$ GFP-expressing mice.

For something like this, just try your best to describe what it is. I don't think there's a definitive way to do this.

For Cx3cr1 +/GFP +/+:

"Cx3cr1 heterozygous with GFP expression" is the most accurate.
"Hetero GFP expression" works if it's clear you're referring to
Cx3cr1.

Cnr1 flox/flox +/- +/-:

"Cnr1 floxed" or "homozygous for floxed Cnr1" are both good.

Cnr1 flox/+ +/- :

"Cnr1 heterozygous floxed" works well.

Cx3cr1 CreER/CreER +/+ +/+:

"Cx3cr1 CreER homozygous" is cleanest.

Cx3cr1 CreER/+ +/- :

"Cx3cr1 CreER heterozygous" is perfect.

CreER is a chimeric protein combining Cre recombinase with a mutated estrogen receptor domain. This makes it activatable by tamoxifen administration.

Can you please explain the notation you used in these? For example: the "+/- +/-" in "Cnr1 flox/flox +/- +/-."

Thank you for your answer by the way! It was very helpful. I am reading them now from left to right and adding heterozygous or homozygous on the end accordingly.


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Modeling and Predicting Squirrel Populations
I was recommended to come here from the MathSE site. Here is the question:
I am interested in modeling the growth of the Eastern Fox squirrel population in Los Angeles, not only in terms of numbers but also in overall territory. The site iNaturalist has a wonderful database of sitings at my disposal, but I am unfamiliar with the mathematical model(s) that I'd need to use to model and predict their growth and territory.
I have taken a quick look at a paper on regular migration and this BiologySE post. Would this be what I am looking for? Or does the question I'm asking require something else? Thank you in advance for the help.
While the eastern fox squirrel is not migratory, I assume the models from the migration paper can be abstracted for my purposes. Perhaps I am wrong.
Some more information as requested by a commenter:
I am completely new to the theory of population biology. The only data I have is from the iNaturalist site, which has geotags of sitings and # of sitings. I am interested in finding out how the Eastern Fox squirrel population has grown over the past few decades, and how their territory shapes up to that of the native Western Gray squirrel. There has been commentary in Washington and NorCal about the shrinking populations of the latter, but I haven't found anything for Los Angeles. This is why I am asking here -- to see whether Eastern Fox squirrels outnumber Western Grays in a significant way (don't take 'significant' in a technical sense).

Welcome to the Biology SE. Please take the [tour] and visit the [help] for more information on this site and how it works. Lets get some things cleared up - please edit the answers into your question. How much population biology (theoretical or otherwise) do you know? How much do you know about the population as it stands now - what sort of data do you have? What do you want to find out? Also, are they migratory? if not, why do you think a paper on migration will help?

Cross-posted: https://biology.stackexchange.com/q/114138/61455, https://math.stackexchange.com/q/4866740/14578.  Please do not post the same question on multiple sites.

You might try to start with log growth, then use iNaturalist data to see if there are geographical areas of overlap in Eastern Fox and Western Gray sightings. If so, check out Lotka-Volterra to assess the competitive interactions.


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The split in Boroeutheria into Euarchontoglires and Laurasiatheria. Was this due to the opening up of the Atlantic Ocean?
I'm asking this question as a cat owner. I've seen a figure like 94 MA for the most recent ancestor of both cats and humans (this from, I guess, molecular clock arguments), and it kind of lines up with the geologic timescale of the Atlantic ocean opening up south to north. Moreover, Laurasiatheria literally means "beasts from Laurasia", and Boreoeutheria is "true northern beasts".

I'm starting to think I'm asking this question in the wrong community. Maybe geology is closer to home?

I disagree, this is the right site for phylogeny questions.

It would seem the answer is "No" at the current understanding of this issue.
You should understand that this is a very complex topic, which is based on both molecular (i.e. DNA sequences or protein sequences derived from DNA or by protein sequencing) and morphology. These are both hamstrung to some extent by the lack of mammalian fossils from this period to confirm any tree, but there are a fair number of early mammalian fossils for this period in particularly in North America but also from Eurasia. Most of the implied phylogeny analyses have been done using DNA from existing species and these results don't always agree depending on which sequences are used. To quote one paper from 20081, where segments are the different DNA sequences they analyzed:

Laurasiatheria was generally not well resolved in any of our analyses, and orders within Laurasiatheria did not have a consistent relationship among the different segments.

It would seem that the Laurasiatheria2 and the Euarchontoglires3 are fairly widely supported as "real" clades, both based on DNA sequences, but there is some dispute as to whether Boreoeutheria is real, with some saying yes, others no.
A 2007 paper from Wildman et al., 20074, had this to say:

The mode of diversification between Laurasiatheria and Euarchontoglires remains murky, and it is unclear whether this was primarily because of vicariance between North America and Eurasia, some other vicariant event, or dispersal.

I found a paper by Springer et al. from 2011, which seems to be the current authority on the question, and is free to read on PubMed Central (see last link in reference; it might also be free to read at the DOI link, which takes you to the publication in the Philosophical Transactions journal)5.
In the section titled Placental Biogeography, they state (with my emphasis), that Eurasia, not North America is the likely origin:

The remaining placental orders are placed in Laurasiatheria (Eulipotyphla, Chiroptera, Perissodactyla, Cetartiodactyla, Carnivora, Pholidota) and Euarchontoglires (Primates, Dermoptera, Scandentia, Rodentia, Lagomorpha). With the exception of bats, these orders have first fossil occurrences that are exclusively Laurasian. Our reconstructions provide support for Eurasia, but not North America, as the ancestral area for these clades...
...Further, the fossil record suggests that Eutheria were dominant in Eurasia throughout the Cretaceous, but were absent from North America through much of the Late Cretaceous and only attained appreciable diversity there during the last approximately 10 Myr of the period

More recent papers seem to take this information as understood and I can't see any more recent papers that dispute it.
Refs:

Prasad AB, Allard MW; NISC Comparative Sequencing Program; Green ED. Confirming the phylogeny of mammals by use of large comparative sequence data sets. Mol Biol Evol. 2008 Sep;25(9):1795-808. doi: https://doi.org/10.1093/molbev/msn104. Epub 2008 May 2. PMID: 18453548; PMCID: PMC2515873.

Waddell PJ, Okada N, Hasegawa M. Towards resolving the interordinal relationships of placental mammals. Syst Biol. 1999 Mar;48(1):1-5. PMID: 12078634.

Murphy WJ, Eizirik E, Johnson WE, Zhang YP, Ryder OA, O'Brien SJ. Molecular phylogenetics and the origins of placental mammals. Nature. 2001 Feb 1;409(6820):614-8. doi: https://doi.org/10.1038/35054550. PMID: 11214319.

Wildman DE, Uddin M, Opazo JC, Liu G, Lefort V, Guindon S, Gascuel O, Grossman LI, Romero R, Goodman M. Genomics, biogeography, and the diversification of placental mammals. Proc Natl Acad Sci U S A. 2007 Sep 4;104(36):14395-400. doi: https://doi.org/10.1073/pnas.0704342104. Epub 2007 Aug 29. PMID: 17728403; PMCID: PMC1958817.

Springer MS, Meredith RW, Janecka JE, Murphy WJ. The historical biogeography of Mammalia. Philos Trans R Soc Lond B Biol Sci. 2011 Sep 12;366(1577):2478-502. doi: https://doi.org/10.1098/rstb.2011.0023. PMID: 21807730; PMCID: PMC3138613.


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Patterns of craniofacial topology
There are animals which are not directly related but have similar craniofacial topology.
For example: koala, donkey, mouse, turtle, parrot all have features seemingly belonging to same topology, especially by baby species.
Mostly it is features like bridge, bridge to eye socket position, nostrils, upper jaw.
Here is a sketch of what I see as a pattern:

Pronounced bridge, eye sockets on the same centerline with the bridge base. Nostrils and upper jaw center area.
Interesting is that there are other "groups" of species that also seem to have their own pattern.
E.g. cats and owls.
It appears to me that there are several basic patterns that can be observed through many species, and thus
species can be classified in such way.
Are there studies of craniofacial topologies that have connections with patterns and such classifications?

Can you be more clear about what you mean? Similar compared to what?

If it's not a coincidence (which is correct) and you don't think evolution is the cause, what do you think is the reason mammals (kind of) look alike? (Personally, deer and koalas don't resemble each other very much to me, but if you mean, hair, bilateral symmetry, eyes, noses, ears, upper and lower jaws, etc., then yes, I agree.)

You may be interested in Paedomorphosis

@BryanKrause some clarifications are added

I'm still not really clear what you think these species have in common that other related species do not.

The morphogenesis of craniofacial structures is a rather elaborately controlled process at both the molecular and mechanical levels. There are a variety of papers on the subject that you might consult. While not an expert, I suspect that this question is too broad to adequately answer all at once.

@BryanKrause that would require comparing at least two types and adding more images. I'll add it later.


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Can a school of 'suction cup' fish block the gills of a predator as a defence mechanism?
(This is about a FICTIONAL FISH - I MADE UP THE FISH. To my knowledge, this concept is not based on any real principles or precedents.)
Now, this fish has a strong capability for suction, and very little in the way of offensive capabilites. As such, I had the idea that a swarm of these fish could use their suction to attach themselves to the gills of predators (i.e. sort of 'stapling' themselves to the space around the gill using suction, although I can include gripping teeth if necessary), thereby starving the predator of oxygen and either
My fish is about 25cm/10 inches long. It is based on the extinct Sacabambaspis (see picture below), and I imagine the small mouth would make effective suction easier.

Is this realistic or possible?
Sorry if this doesn't fit Biology Stack Exchange, I can put it up on Worldbuilding SE if it's not suited. I couldn't find anything about made up creatures in the rules.

Since you're not asking about something in the world, perhaps your fictional creature might find a better home on Worldbuilding as you suggest. We don't do hypotheticals so much here.

If you look at the “rules” they exclude questions that can only be answered by opinions, rather than facts. Indeed this is one option in the close vote form, which you will probably be unaware of. It should, however be obvious from the examples in the [Tour].

Surely a hypothetical question can have a definitive, non-opinion answer (not sure if this one does, though).

I will give you some ideas to think about. I think your hypothetical is plausible enough.

Most fish gills are protected by an operculum (bony gill cover). This might make attaching directly to the gill filaments difficult.
If they rely solely on suction, the water flow could dislodge them, especially if the fish can thrash. Tooth-like structures may be a good idea.

The question asks if this is realistic or possible. I think so. I see no obvious reasons why this would be impossible. Though, in reality, the prey of these suction fish would eventually evolve defense mechanisms.


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Best way to predict the effects of deletion mutations on protein function?
I have the coding sequences of a WT gene and several mutants of this gene (deletion mutations varying from 5bp to 50% of the sequence deleted). What is the best method for inferring the impact of these mutations on the final protein?
I am looking at AlphaFold but papers have found it is no good at predicting protein stability change (ΔΔG) based on point mutations, and AlphaFold answer one of their FAQs by saying "AlphaFold has not been validated for predicting the effect of mutations".
So... what is the best method for predicting effects of mutations based on sequences?

Not sure, but look into Rosetta or DynaMut. Obviously, actual experimental validation is the best. Are there known deletions in related species or the same species (ExAC, gnomAD, ClinVar)?

I would suggest looking at the sequence conservation of the protein domains / sequence positions of interest.
One such tool that is oriented towards human genomics is CADD. This tool can provide a score predicting how damaging a mutation at the position in question might be.
But in the absence of a convenient interface, you could generate a sequence alignment yourself of homologous proteins and compute the conservation (similarity or identity) of the position across such homologous proteins.
Unfortunately I am not sure that these methods will make you feel much wiser, they will largely be useful for saying that one area of a protein is more important than another, they will not necessarily tell you much about the functional changes in the protein.
It is of course always a good idea to look at the domain annotations of the protein in question, e.g. mutations in the active site of an enzyme may affect activity, mutations in a DNA binding domain are more likely to affect DNA affinity, etc.
Uniprot is one place where you can investigate both of these at once to some extent.


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Is this contamination, debris?
I have been culturing SVG cell lines. This line was obtained as a live culture from another institute and was delivered by mail. After subculturing , I saw some particles, and I do not know what they are. The cells are growing well, no debris, no colour change, no foul smell. Can anybody tell me what this is?

Welcome JEME. Could you [edit] to add some details like: 1) The magnification (or at least some idea of scale), 2) Which objects are you referring to, do you mean the crinkled object in the centre of field or something else?

It is an imperfection in your plasticware.

Is it? Assuming it's the large object in the centre of the view, it looks like it is above the focal plane but still close to focus, which would indicate something other than plasticware - perhaps debris from the plasticware, or something like a protein precipitate from FBS


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With CRISPR-mediated gene editing, can the PAM for the guide RNA also be site of the desired edit?
I'm hoping to use CRISPR-Cas9 in yeast to change a single nucleotide in a gene of interest. While there are a couple potential guide RNA sequences I could use, the best option's PAM sequence (TGG) it contains the site of my desired edit (TGG > TTG). Would there be any issue with this?


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Membrane Potential Question

Hello, I've come across this question and am slightly confused. The question states that for this scenario the membrane is only permeable to K+, thus would the membrane potential in this state just be the equilibrium potential of K+? If this is not the case I would not know how to calculate the resting potential. I've tried finding the individual equilibrium potential of the ions and finding the potential difference but this gives a value that would not be correct given the circumstances in the question. Anyone have any idea how to proceed?

Please do not post text as graphics. It discriminates against those with visual problems — especially those using screen readers — and it prevents questions being indexed.

Usually we use the Goldman Equation when calculating membrane potential with multiple ions and the Nernst equation for calculating reversal potentials for individual ions.
Try out the Goldman Equation with all these ions, setting the permeability of the ones that are zero to zero. Try the Nernst equation for potassium. You'll find that when you set the permeability to zero of all ions but one the Goldman Equation algebraically becomes the same as the Nernst equation for a single ion.
https://en.m.wikipedia.org/wiki/Goldman_equation
https://en.m.wikipedia.org/wiki/Nernst_equation


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Preserving a sample for mtDNA and nuclear DNA analysis
Imagine a person in the early half of the 20th century (1900 to 1950) took some kind of sample(s) from a living human body using any technology of the era.  They then stored it using any technology available, but could not use prolonged (70+ years) refrigeration.
We want to be able to do useful analysis of that sample in the year 2024.  We particularly might want to be able to sequence mtDNA and nucleus DNA, look at telomeres and methylation.  Furthermore, we especially want to be able to try to determine the age of the individual, and even better, from what approximate generation they were (i.e. plus/minus a hundred years).
Is such sample storage even possible?
Using my "just enough knowledge to ask stupid questions":

Could a sample be preserved in formalin and then immersed in alcohol, all in an air-free sealed container, and preserve the needed DNA strands and/or other biochemistry?
Could ethanol precipitation of DNA be used on the sample
contemporaneously, and that precipitate instead be stored safely for
70+ years?

Maybe these other questions apply?

DIY extraction and long term storage of human DNA sample?
How realistic is to use DNA for long term storage?
Isopropanol precipitation of DNA - duration and magnitude of cold storage

(I'm an old hand at Stack Overflow / Stack Exchange sites, but this is my first post to Biology.  Gentle feedback appreciated.)

If you're wanting to store DNA long-term at room temperature, you'll probably want to dry it. DNA extraction methods have been known since 1869. You can then use ethanol to precipitate the DNA, centrifuge it, and dry off the ethanol. The dried DNA pellet is surprisingly stable (though still maybe not great at this time scale, depending on what you want to do with it). Adsorbing the DNA onto a matrix, including silk, can increase preservation.
You could also try preserving the tissue itself. Two good options would be freeze drying the tissue, or preserving it in 95% ethanol. You would not want to store in formalin, as that creates DNA fragmentation and cross-linkages.

Thank you so much!  That's exactly the kind of knowledge I was looking for!

For anyone curious, @Darlingtonia's answer is what I needed.  It also gave me the search terms that Google could use to finally find further useful information.  In particular, a paper published at the National Center for Biotechnology Information, linked here.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3910151/
First paragraph of the abstract:

The globalization of DNA barcoding will require core analytical facilities to develop cost-effective, efficient protocols for the shipment and archival storage of DNA extracts and PCR products. We evaluated three dry-state DNA stabilization systems: commercial Biomatrica® DNAstable® plates, home-made trehalose and polyvinyl alcohol (PVA) plates on 96-well panels of insect DNA stored at 56 °C and at room temperature. Controls included unprotected samples that were stored dry at room temperature and at 56 °C, and diluted samples held at 4 °C and at −20 °C. PCR and selective sequencing were performed over a 4-year interval to test the condition of DNA extracts. Biomatrica® provided better protection of DNA at 56 °C and at room temperature than trehalose and PVA, especially for diluted samples.


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What is the difference between "microproteome" and "peptidome"?
I know that "peptide" is sometimes used to refer to both microproteins and short peptides derived from longer proteins (post-translational), but I do not know whether this extends to "peptidome" versus "microproteome"?


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Why do old willows have so many broken limbs?
Willows have a reputation for being very flexible. In a nearby park (PA), where I often walk, there are about 20 willows growing by a stream. Willows over 30' to 40' tall have almost all their major limbs broken off near the trunk, why is this?
Do willows become brittle as they age?

I took a recent survey:
All willows over about 30" diameter trunk were severely damaged.
Willows up to about 24" diameter trunk showed no damage, even though some are now taller than those older trees.
Those in between showed little damage.
Some of those damaged willows show heavy infestation of wood boring insects on their trunks and damaged limbs.

Welcome to the Biology Stack Exchange. Please take the [tour] and visit the [help] for more information on this site and how it works. We would need a little more information on these trees - what species of willow are they? I can see 8 species listed for Pennsylvania. Are the breaks recent (i.e. associated with a weather event - storms and droughts can both cause this sort of damage)? Are the breaks all of a similar age or different ages? A picture might help.

@bob1 Thanks for your comment. I added a picture to my question and some other observations. I do not know which species they are, though younger and older trees seem to me to be the same species. The recent heavy wet snow caused one of the infested trees to fall, and large limbs regularly break off during strong winds and storms.

These trees appear to be Salix x fragilis AKA crack willow or brittle willow. The form is more upright and taller than the common weeping willow, but has drooping pendulous thin branches on the lower limbs. We would need to see the leaves for confirmation of species.
These are quite variable in form and are an introduced species in the USA, having origins in Europe and Asia as a cross between two different species, S. alba and S. euxina.
As the name suggests, the tree is fragile and breaks easily from storm, ice, snow etc.  From the common height of break off and similar diameters of the trees, I suspect that they are similar in age and were hit several years ago by a single weather event.
Given your description of the changes in trunk diameter and younger (smaller trunk) trees not showing damage, I would hazard an event probably well over 20 years ago - perhaps the Blizzards of '93 or '96.

so in conclusion, you would say that willow trees do not become more fragile as they age. The group of willow I observed were Salix-x-fragilis "crack willows" which are a very fragile species of willow and most likely were all damaged in a severe storm perhaps 30 years ago.

@Rich Yes, that's about it. I don't think they become more fragile with age, but I guess a young branch might be a bit more flexible so less likely to fracture?


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CD45 as a microglial marker in Fluorescence-Activated Cell Sorting (FACS)
I am reading this article: Microglial cannabinoid receptor type 1 mediates social memory deficits in mice produced by adolescent THC exposure and 16p11.2 duplication.
In Figure 1a, the authors are using Fluorescence-Activated Cell Sorting (FACS) and are identifying different cell types as follows: microglia (CD45$^+$CD11b$^+$TMEM119$^+$), astrocyte (ACSA-2$^+$), and neuron (NeuN$^+$).

My understanding is that the upper and lower panels indicate two different batches of cells. The upper panel shows neurons identified by the NeuN marker. At first I was thinking that because of this "microglia (CD45$^+$CD11b$^+$TMEM119$^+$)," that cells were selected from Q2 in the left lower panel and were further examined in the context of expressing TMEM119 and ACSA-2 to differentiate between microglia and astrocytes.
The problem I am running into with my interpretation is two-fold. Reading from abcam: "A combination of CD11b and CD45 labeling can be used to distinguish microglia from macrophages. Resting microglia are CD11b$^{hi}$, CD45$^{low}$, whereas macrophages are CD11b$^{hi}$, CD45$^{hi}$." This would seem to indicate that Q1 is the pool of microglia. As far as Q2, if it represents macrophages, why would there be a larger presence than the resident immune cell microglia?
The second issue with my interpretation is that I do not see any literature suggesting that astrocytes express CD11b or CD45. Any help here is greatly appreciated!
Edit: Regarding the abcam source, they also provide this with references.
Edit2: "In order to confirm Cnr1 expression at the protein level, we used magnetic activated cell sorting (MACS) to isolate microglia-enriched CD11b$^+$ cells, ACSA-2$^+$ astrocytes, and remaining cells including neurons from the cerebral cortex of Cx3cr1$^{CreER/+}$;Cnr1$^{flox/flox}$ mice and littermate controls (Cx3cr1$^{CreER/+}$;Cnr1$^{+/+}$). We used this approach as it produces a higher cell yield than FACS with over 95% of collected CD11b$^{+}$ cells being microglia." I feel like this excerpt from the paper should help to provide some clarification to whoever answers this question.


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Why do we choose to use agar instead of agarose in various microbial applications?
When performing gel-electrophoresis we always use agarose. Is there a reason we can't perform it using Agar?
And in microbial culture Agar is commonly used as solidifying agent, could this be replaced with agarose.
I understand the difference in price, composition, and manufacturing but other than that how would it affect the result?

Welcome to the Biology Stack Exchange. Please visit the [help] for more information on this site and how it works. Please also note we have a homework requirement. You could help your question  by telling us what you have found out.

Agarose is indeed more expensive than agar because it is purified from agar, i.e. it is agar minus the agaropectin. Agaropectin is heterogeneous, and modified with acidic side-groups. The consequence of this is that it would interfere with the migration of nucleic acid in the gel.
Similarly, agarose is sometimes used for observing zones of inhibitions of growth or lysis plages of microorganisms caused some antimicrobials, because it interferes less with their diffusion than agar.


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What are ways to measure carbon fixation of microalgae?
So I am conducting a very simple experiment about microalgaes, particularly their biofixation of carbon dioxide. In a small compartment, where microalgaes are suspended in water with an air pump that supplies carbon dioxide for growth and development.

What are possible techniques to measure the biofixation rate that utilizes less highly specialized equipment as much as possible?
Is it also possible to conduct gravimetric analysis to measure biofixation?


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Pedigree Analysis
I was doing some pedigree work and couldn't figure out why my answer is wrong. This chart is supposed to be an autosomal Dominant method of inheritance, but I am finding an autosomal recessive pattern that fits? If anyone can point out my mistake, that would be appreciated. I have labelled my attempt above each member, where aa = disease phenotype and Aa or AA is a normal phenotype. The disease pattern describes Marfan syndrome (autosomal dominant). Any help or pointers would be appreciated.
I decided not to label the pt who has a *'s family, because there are several possibilities that don't affect the answer.
Edit: I also found an autosomal dominant pattern that fits, but the issue is with the autosomal recessive pattern, so I am only posting that one.

Is it reasonable to assume that everybody that enters/"marries into" this family tree happens to be a carrier for the same recessive trait?

I do not see any "error" in the logic of your pedigree.
However, pedigree analysis is not a logic puzzle; it's a practical tool. There are additional logical solutions you have not come up with. For example, every affected individual in this tree could result from a de novo somatic mutation with no inheritance at all. Or the phenotype could be not genetic at all: the observed pedigree pattern might well apply to "people who have been in a serious car accident". Or, it could be a polygenic trait that is not easily tracked in pedigrees even if there is some underlying inheritance.
You're expected to have some additional assumptions that are met to even bother undertaking a pedigree analysis, and one of those likely assumptions is that the disease is rare. That is, it's rare enough to rule out more ordinary polygenic traits and make you think there's a single gene involved. In that case, it is not reasonable to assume that everyone entering the pedigree from outside (e.g. marrying in/breeding in) happens to be a carrier of the disease.
It looks like that's what was necessary to make this tree work: besides the initial aa/Aa pairing at the top, there are 4 other individuals that come from outside and pair with someone who is a descendent of that pairing (there are actually 5 in a sense, if you include both of *'s parents, but we can reduce it to just * and count them as #4). You've marked all 4 of these as heterozygous carriers for this pedigree to work. It would be extremely unlikely for this to happen with a rare disease.
See also the concept of Occam's razor.

Bryan already answered in a very exhaustive and elegant way. The fact is that most textbooks and teachers, when dealing with recessive traits, assume they are severe diseases, for which it is reasonable to expect that they are rare in the population. This will also make the recessive allele rare in the population, and the likelihood that 4 married-in all carry the disease allele is so low to be neglectable.


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Plant tissue cultures without Phytohormones?
Is it possible to do tissue cultures without using phytohormones like BAP or NAA. So to just use for instance MS-Media.
Online, I just found that every plant is possible to culture with the right media and growth regulators. But I want to know if it is possible in theory to do tissue cultures without using hormones and if it is what are the disadvantages of that.


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Can DNA reveal an approximate chronological generation of a person?
Given complete nuclear and mitochondrial DNA from an individual, would it be possible to approximate in what generation they were born?
For example, imagine DNA from someone who lived circa 200 to 700 years ago somewhere in Alsace (modern France).  Could we narrow that range to say they were likely born between 400 and 500 years ago using DNA evidence?
Imagine this is an expense is no object project with a 1 to 3 year period starting in 2024 for doing the work.

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It is possible w/ population history and local admixture patterns or new haplotypes appearing in addition to mutations, but is not a defined or accepted technique and likely to be inaccurate.

Briefly, no.
There may be an argument for using similarity to previously observed archaic hominin genomes that you could impute the age by similarity to e.g. Neanderthals. But those would be very old samples.
There's maybe an argument that you could look at the level of admixture in the sample and try to guess where it happened in the human population expansion, or by relating it to pedigrees. My gut sense is that it would be very difficult to differentiate it from currently existing humans.
That hasn't stopped people from trying; I haven't read the relevant paper in detail but it seems to only work in judging relative dates of somewhat closely related people, e.g. "this skeleton probably isn't the same date as this other skeleton found in the same location". It isn't an absolute measure of date, and it requires reference to some other sample. The authors themselves say it isn't a replacement for radiocarbon dating.
But in practice no one bothers, given that ascertainment of ancient hominins is so low. It is much simpler to just radiocarbon date the same sample that you sequence, even if you are already sequencing. Radiocarbon dating seems to have pretty good resolution in that age range, so you might as well just do that.
Methylated cytosines are approximately as stable and unmethylated cytosines in ancient DNA, so they aren't a good measure.


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PCR: Are primers designed to be complementary to a DNA sequence within the template DNA or to the 3' ends of the template DNA?
I was told that in order to produce primers, the DNA sequences at the 3' end of the template strand must be known, and that these primers are complementary to 3' ends of the template DNA.
However, that causes me confusion when I view the following image from my college class, which show that the primers anneal to the template DNA further down the strand:

So do the primers need to be complementary to the last 20-35 bases at the 3' ends of the template DNA or simply to any portion of 20-35 bases near the 3' end?

You seem to be confused about the template DNA having a relevant "end".  Most DNA molecules one would start with would be very long, often an entire chromosome.  The 3' ends of the two strands are essentially never near the piece one is trying to amplify with PCR.  So, no, the primers do not match these ends.
For PCR, you are trying to amplify a "region of interest" within the millions of bases you start with.  The region is necessarily bounded by two known sequences (otherwise you have nothing to prime the DNA replication).  The two known sequences define the region of interest and are the sequences one creates the two (different) primers -- the green and orange primers shown in the question's diagram. Since those two primers define the region of interest, and DNA replication will proceed in one direction (from 3' to 5'), they are exactly the two ends of the region to be replicated.  These primers of course anneal very, very far from the ends of the chromosomes.


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Cell Size and Morphology of Microalgae under Wastewater
Hypothetical situation ahead, I am just curious whether the cell size and cell morphology of microalgae changes when it is under the influence of wastewater. For instance, mixing the microalgae culture with wastewater. If if does happens, what potential reasons are the causes of such changes?

Welcome Kris. This is very lacking in detail at present. Please take our [tour] and refer to the [ask] section of the help centre, then you can [edit] to add details such as: 1) Specifically what you mean by wastewater and in what concentration. 2) What microalgae are you referring to and what's its natural environment. 3) To show your own research into the question as per our requirements (see the homework policy section for details).


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What is the inflorescence of Solanaceae family?

Inflorescence of Solanaceae : Solitary, Axillary or Cymose as in solanum

This was written in the textbook I was studying. Doesn't Solitary encompasses axillary inflorescence as well ?

Welcome Arun. Please take our [tour] and refer to the [help] particularly the [ask] section. You'll note that we deal with single focused questions, one per post. At present your question is very broad as the family you refer to has many different members (thousands of species) with different morphology. Could you [edit] to be much more specific and to show your prior research.

Sorry for causing inconvenience.  It is written in my textbook that inflorescence of Solanaceae family is solitary, axillary or cymose. That's the reason I asked the 2nd question because as far as I know, axillary are also a type of solitary inflorescence . Hope I will get some help.


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What is the word for a small (under 50 bp) structural variant?
What is the word for a small (under 50 bp) SV? Perhaps it is MNV (multi-nucleotide variant)? Something like microindel? I do not like SNV as "single nucleotide" is not really correct and I want a indel-specific term.
SV = structural variant
bp = base pairs
SNV = single nucleotide variant
indel = insertion or deletion

@JiminyCricket. Yes, done

One can clearly find instances of "microindel" in the literature. However it does not seem to be very common.
"Small indel" appears to be the preferred term, noted here:

Typically, small indels are termed for insertions/deletions of shorter than 50 bps while longer ones are considered as structural variants (SVs) [2, 3].

For example, DeepVariant uses this term.


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Near-infrared EM spectrum and uses in biology (specifically in botany/vegetation)
A little explanation for my problem:
I have a large dataset - images of aerial scans of my country. These images are available in both RGB and RGBN colors - N specifying the near-infrared color channel. I plan to  use this data/images to make a computer-simulated realistic landscape game. However, the data has a resolution of around 15cm, which means I can't really expect realistic depictions of individual blades of grass. I will need some kind of interpolation.
I could manage pretty well with the RGB colors, but I would like to know some uses of the near-infrared color channel for this kind of problem - which is analysing vegetation, ground, etc. - and how could it help me with this kind of project.
I did try doing some research: I found this neat little picture that illustrates how we can analyze if a plant is healthy or not.

Green vegetation (left) absorbs visible light and reflects near-infrared light; Sparse vegetation (right) reflects more visible light and less near-infrared light. (source)

Thank you for any help.

Sorry, I can't see what your question is here - are you looking for data on how to resolve types of plants based on NIR or something else? The GIS Stack might be more help. There's also some info in the original publication for your image here, which you incidentally should attribute correctly!

To be fair, it's a very general question. I am looking for a botanist's view on this - I am not very well versed in botany nor biology. I'm just looking for some resources as to how to approach this issue. The GIS StackExchange users are not biologists - I believe I'd be redirected to the Biology StackExchange and so on again.

There's quite a bit of information on this topic around - try this search at Pubmed


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Question on evolution theory from Book:Origin of species
Why, if species have descended from other species by insensibly fine gradations, do we not everywhere see innumerable transitional forms? Why is not all nature in confusion instead of the species being, as we see them, well defined? — On the Origin of Species, chapter 6, published 1859.
Is there any good explanation to question by any biologist ?

What makes you think species are well defined? There are numerous questions here where people are confused by how lacking species definitions are and how often they don't make sense as strict categories. All because evolution means those strict categories don't make sense.

What also makes you think you aren't seeing transitional forms everywhere right now? If, as you say, evolution takes place over insensible gradations, how long do you think this takes, and what forms are you expecting to see in a given species - and what do you have to compare it to, to see the transitions? Do you think the transitional forms are all easily visible?

A well-known example of this would be chichlids in the African Great Lakes, see also Darwin's finches for one that the man himself noticed, but didn't know how to interpret what he was seeing at the time.

Maybe you think that "transitional species" would be birds or bats with half-wings, spiders with 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 legs? What is your concept of transitional species? I don't know what you mean by that, so it's hard to answer, but I do think (if it's the above) it shows a foundational misunderstanding of evolution and that ever-so-important selection pressure.

We do see transitional forms, and species are not well defined.

Why, if species have descended from other species by insensibly fine
gradations, do we not everywhere see innumerable transitional forms?

We don't necessarily see the transitional forms, but they are everywhere non the less. Invisible transitional forms might take the form of increased intelligence  or increased resistance to disease and numerous other beneficial traits that are not readily apparent on superficial observation.
The human race itself is an example of a species which is full of graduations that are readily visible to the human eye such as different, eye, hair and skin colours, different adult heights, different hair texture, different morphologies etc, etc. Dogs possibly have even greater diversity for a single species.
A problem is how we define a species. One definition of separate species is that they are unable to cross breed, but this is not strictly adhered to and difficult in practice to determine for moral reasons. One example is a few years ago the forest elephant was declared a distinct species from the African elephant and therefore deserved conservation funding in its own right. They argued it was a distinct species because they are smaller on average and preferred living in the forest unlike the African elephant. This is silly because by the same criteria, bush pygmies would be a different species which of course they are not. I suspect the definition in the forest elephant case was for funding reasons.

Why is not all nature in confusion instead of the species being, as we
see them, well defined?

Some species appear to be well defined from a superficial visual observation, such as penguins. I suspect this is due to sexual selection pressures. The females probably have a gene that gives them a preference for a mate of standard penguin shape and size and this drives a kind of uniformity, but beneath the surface the invisible evolutionary changes are going on such as increased resistance to disease, increased fertility, better fish catching skills, better ability to absorb and process nutrients and energy from the fish they do catch, increased resistance to cold and so on.
In a large population of a species, there is a lot of mixing of the traits going on, both visible and invisible but because of the shared gene pool the population generally evolves in the same direction as an averaged whole. However if a portion of the population gets cuts off from the the larger population (eg separation of land masses a long time ago) then the two populations can diverge due to different selection pressures and sometimes due to a disproportionate prevalence of a particular gene in the founder population due to statistical chance.

See Wikipedia divergent evolution.


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Alzheimer's drug Aduhelm causing viral encephalitis
Over 40% of patients who received Biogen's anti-amyloid beta drug Aduhelm had "brain swelling or bleeding." (See, https://jamanetwork.com/journals/jamainternalmedicine/fullarticle/2792897) Could these reports of swelling/bleeding include viral encephalitis? I haven't been able to find any sources indicating that this is what patients had, but there is reason to believe that removing amyloid beta from the brains of Alzheimer's patients could increase viral encephalitis (see, https://www.cell.com/neuron/pdf/S0896-6273(18)30526-9.pdf). Is there a reason that the clinical trial monitors/sponsors would not have to publicly report it as such?

Why do you think it is viral encephalitis?

Hey @Chris, thanks for your message. The second link is to a paper published in Cell - it shows that when herpes simplex virus 1 (HSV1) infects a transgenic Alzheimer's Disease mouse model, the infection leads to increased amyloid beta deposits. These increased amyloid beta deposits (specifically Aβ42 oligomers) protect mouse models from herpesvirus encephalitis and inhibit several types of herpesviruses. This indicates that when Aduhelm clears amyloid plaques from the brain, it could inadvertently let herpesviruses go wild...

... Many papers have shown that herpesviruses, including HSV1, HHV-6A, and HHV7 (and others) are consistently found in the brain, specifically in the parts of the brain most ravaged by Alzheimer's Disease (see, Readhead et al., 2018 - https://www.cell.com/neuron/pdf/S0896-6273(18)30421-5.pdf)

I suggest that SE Medical Sciences would be a better fit for this question. The examples of appropriate questions for this site in the [Tour] do not suggest clinical trials or disease are part of the SE Biology remit.

This is a very interesting question. I think this site is correct for the question because it falls under "questions about the biological mechanisms behind medical conditions."


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How many protons are pumped into the intermembrane space by Complex IV in OXPHOS?
According to Sazanov (2015), "to reduce oxygen to water, four 'chemical' protons are taken from the matrix side [...]. In addition, four protons are pumped across the membrane into the IMS". A similar statement is made in the review by Vercellino & Sazanov (2022, pp. 152–3).
How is this compatible with only 10 protons being pumped into the IMS in total and only 2 by Complex IV (see also How many protons are pumped out per pair of electrons from NADH in oxidative phosphorylation?)? Does the number of 10 perhaps simply result from the assumption of a 10/3 H+/ATP ratio? What am I missing here?
(the literature on H+/2e- stoichiometry, if anything, added to my confusion)

*10 H+ per molecule NADH


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Does the VR headsets make us focus extremely close?
Firstly, if I ask this question in Ask Different or Super User, the users there may say this is related to a biology or physics.
The background is: we were in general told that: don't read a book too close, or don't watch the TV too close, because it will make our eyes focus with a really short focal length, and that means our eye muscle has to make the biological lens in our eyes to be extremely convex shaped.  As a result, we can easily develop myopia this way.
Does this apply to VR headsets, such as Apple's Vision Pro, or Meta's Quest 3? In these cases, the "display" is like one or two centimeters or fractions of an inch away from our eyes.
So in that case, does that make our focal length to be extremely short and therefore, injure our eyes in the long term?

The VR headset is presenting two different images to each eye, so strictly speaking, the eyes are not looking at the same object (as in a book or TV).
This idea has been used in non electronic devices (Stereoscope) for a long time to see pictures in 3D.
So the eyes are not actually focusing at the VR device screen but further away.

I don't think it works this way:  Mom says: hey Peter, don't watch TV so close, like 2 feet away. Peter says: Mom, don't worry, I am closing one eye to watch the TV 2 feet away, so it is not using two eyes and so it doesn't matter

@heracho its about distance to the screen from the eye

So, your question is more about this phenomena? : https://en.wikipedia.org/wiki/Vergence-accommodation_conflict

The display may be two centimetres away, but there are lenses between the display and the eyes, and they project the image to make it look like it is much further way. This is necessary, because most people cannot focus properly on an object that is only 2 cm away, and it would cause the eye strain or fatigue that you mention.
It's a bit like looking through prescription glasses. Lens are very close to our eyes, but it is not the lenses that we focus on. We focus on where the light rays coming through the lenses appear to be coming from.


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Identification of Secondary metabolites gene clusters in fungi
I am trying to identify secondary metabolites gene clusters specifically PKS from endophytes through PCR then sanger sequencing. Are there set of specific primers available or do I have to design my own?

What fungal species is it ? Are there primers in the literature designed by people who study the same secondary metabolites as you ?
What aspect of the gene clusters do you want to study ?


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Is there a program that demonstrates overall fitness will increase in a population overtime?
Of course this will not always be the case with environmental factors, but generally speaking. I'm searching for a program that shows evolution works from a statistical standpoint.

Which theory of evolution requires fitness to increase in populations over time to "work"?

Look into Richard Lenski's long-term experimental evolution work. In addition to more than 60,000 generations of E. coli evolution with numerous fitness published fitness studies, he has published some work trying to study evolution in "digital organisms."

One can readily design such an evolutionary simulation. There are many such programs, many of them designed for educational purposes. Others are used to explore the theoretical effect of varying certain parameters including formalized environmental factors. Is that what you mean?

Yes. Fisher wrote on this (hypersphere). Then it was improved by Kimura. Now, as the other commenters mention, there are modern much better ways of simulating this.


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Can someone identify this species of Protolophus?
I have posted my observation on iNaturalist, but so far this Harvestman has only been identified as genus, Protolophus. Here are three angles of the Harvestman in question, which has been observed in North Sacramento County, California.


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Evolution of the human Y chromosome
This recent news article suggests the number of genes in human male Y chromosome has steadily been reducing in the Y chromosome for a long time and is in danger of dying out all together.
What I want to know, is if this is a bad thing, why don't normal evolutionary forces act to prevent this? For example if some individual is born in the future with a further reduced gene count in their Y chromosome and this reduces or eliminates their fertility, they will will have less offspring and that trait will die out. Why would the human race evolve in a way that is disadvantages to reproduction, when that is the main driving force of evolution?

What I want to know, is if this is a bad thing, why don't normal evolutionary forces act to prevent this?

You seem to be attributing a moral value to an evolutionary process. So does the article you linked to your post (probably for sensationalism), which is not a scientific way of reasoning. I suggest you read the original article instead. In the abstract, you can read :

The results of our study suggest these forces are not exclusive to the Y chromosome, and chromosomal degeneration may have occurred throughout our evolutionary history. The reduction of recombination could additionally result in rapid fixation through isolation, of specialized functions resulting in a cost-benefit relationship during times of intense selective pressure.

Therefore, this trend is not specific to the Y chromosome, and there is no evidence that the reduction in the number of genes on the Y chromosome has been associated to a decrease in the fitness of humans.


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What is important in the Luria-Latarjet effect?
In the seminal paper

Luria, S.E. and Latarjet, R. (1964) “Ultraviolet irradiation of
bacteriophage during intracellular growth”. Proceedings of the
National Academy of Sciences of the United States of America, vol. 52,
pp. 1521-1529.

Luria and Latarjet reported that the sensitivity of phages within infected bacteria decreased sharply after about 10 min p.i.
They assumed a "screening hypothesis" that assumed that the viral genomes (sensitive to irradiation) were shielded from UV light by "some ultraviolet-absorbing material" (namely the capsid".
This paper led to the so-called "Luria-Laterjet effect" (e.g., doi: 10.1016/0042-6822(68)90011-1 and doi: 10.1017/s0016672300031499). Thus, it must be a relevant feature in virology.
What is the underlying feature of this effect?
I understand that it is proof of a viral eclipse (virions are formed sometimes after infection, albeit they might still not be infectious). Still, some papers raised the involvement of DNA repair (which is usually not mentioned in modern virology text covering viral replication).
Is there something else that this effect explains?
Thank you

I only see two papers which call this the "Luria-Latarjet effect", both of which are the ones you provided as DOI numbers. Two papers is tiny output for a "feature of virology" or it would be more widely studied. Luria and Latarjet found that during infection the sensitivity to DNA damage decreased and then increased again as new phage was produced. I fail to see any feature that isn't explained by Hyman's work.

Fair enough, I though there was more to it. Thanks

Can I add an addition to the question? IS the L-L effect a proof for the presence of restriction enzymes? Luria made such a preliminary discovery soon after this work with Latarjet but I did not understand if he was talking about the same subject (DOI: 10.1128/jb.64.4.557-569.1952 )...

No, I don't think so - though it could make a good new question. The L-L effect described is due to formation of the viral DNA/RNA - when it is in short lengths it is less susceptible to damage from radiation, which has little to do with REs would be my guess.


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Interpretation of narrow-sense heritability over one (using R/S = h^2)
Here is my data:
Mean height score of the total parental population: 5.2
Mean height score of selected parents (those chosen for breeding due to their higher height): 6.4
Mean height score of the offspring (resulting from the selected parents): 7.8
Selection differential (S) for height:
S = Mean height score of selected parents - Mean height score of the total parental population
S = 6.4 - 5.2 = 1.2
Response to selection (R):
R = 7.8 - 5.2 = 2.6
h^2 = R / S
h^2 = 2.6 / 1.2 = 2.167
I confirmed that the data are not wrong. What is the interpretation of this h^2 value?
I am looking for explanations for why h^2 would be above one.

Check your assumptions.
It looks like you're using a formula that assumes linear, additive variance. Your data suggest a system that does not follow these assumptions, so the equations are not valid for the situation you describe.
See also https://en.m.wikipedia.org/wiki/All_models_are_wrong - much of the difficulty in applying simple mathematical formulae to real world data is in deciding/recognizing when your model is sufficiently wrong that it's no longer useful.

What could explain that? Dominance? I thought of that, but I assumed that would decrease h^2, not increase it

@BigMistake I would start by assuming the trait is simply nonlinear. No reason that things have to be linear in the real world just because it's sometimes convenient to model.

Another possibility would be non-independence of the environment. If there's a systematic environment change that's going to affect population parameters outside of the heritability.

Actually, I think dominance could elevate h^2 above 1. The regressed slope would be above 1

I know these things can bias h^2 estimates, but I'm specifically wondering about what can result in them being above one, not just generally inaccurate

Both reasons I suggested could have this result. It's not that surprising: when assumptions are violated an equation is no longer valid.

How would they increase h^2?

They don't, they make the equation you are using to calculate it invalid.

Exactly. I am looking for why h^2 is calculated to be too high, not why the equation is invalid

Because you're using an invalid equation to calculate it because the assumptions that make the equation hold are not met - that's why it's too high. R is a bigger response than is possible under the model that makes that equation hold.

Assumptions not being met ≠ reason why a particular direction of bias is observed. Assumptions being met = reasons why it is wrong, but ≠ reasons why it is wrong in a specific direction.

R is large. That's it. In another situation with violated assumptions it could be small instead. The direction isn't important what is important is that it's not meaningful. If you tried to calculate how much money you have in the bank by multiplying what you deposit by what you spend you get the wrong answer because that equation is wrong. It's not important to explain why the result is too big or too small, it's just not meaningful to use that equation in that situation.


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Qpcr negative efficiency
Hello Stack Exchange Biology world,
I have struggled with my qPRC results for over 5 months. I have a bunch of DNA samples, and I want to find the quantity of Pseudomonas syringae contained. My issue was the extremely high efficiency (sometimes even 1200%) after the run. So, I decided to make everything from scratch. I used a new set of primers and a new qPCR kit (kapa sybr fast qpcr master mix (2x)), and I switched the sterilized double-distilled water from the water filters in the lab to an ampoule one (to eliminate any contamination).
I incubated the bacteria again. I measured the OD=0.998A, before extracting this DNA (via NucleoSpin Soil, Mini kit for DNA from soil) with the following quality as 193μg/mL, 260/230 = 2,41 & 260/280=1,89, which seems high quality.
For the standard curve, I did dilutions of 1:10, 1:100, 1:1000, and 1:10000 using the proportion of 5μl of DNA with 45μl SDW from the ampoule.
So now I have everything new and a "high quality of DNA" to work with. Yesterday, I attempted to perform a qPCR (SYBR green), and the efficiency was -100%. What should be wrong at this time? Is it something that I can change in my 20μl reaction?
SD Water from ampoule 7μl
QPCR Master Mix 10μl
Primer F 0,3 μl
Primer R 0,3 μl
ROX high 0,4 μl
DNA 2μl

The machine I performed the reactions on is the Thermo QuantStudio 5 Real-Time PCR. Below, I am providing the experimental details as well as the qPCR results.
Thanks in advance for any suggestions to help me fix this issue.

Normally, if your qPCR efficiency was negative 100% I would say your dilutions were made properly just entered into the software in reverse order. But your amplification curves are pretty funky. How are you setting the threshold? And what is the baseline determination?  What about the melt curves? The protocol is strange too, in that it's taking multiple fluorescence readings per cycle. I wonder if that's screwing with the baseline readings. Typically just one measurement is taken after annealing/extension. Have you tried a 2-step protocol (deleting the 79C step)?

@MikeyC Thank you for your response. As I'm learning qPCR, I combined the KAPA SYBR® FAST Master Mix with a P. syringae protocol I found.  https://journals.asm.org/doi/10.1128/aem. The main issue was that the protocol used a different qPCR mix and annealing at 54°C compared to KAPA SYBR®(annealing at 60°C). I adjusted accordingly, but perhaps incorrectly. About the threshold and baseline determination, I used default settings. For my next run, I will only keep step 2 for fluorescence reading as recommended. Any further suggestions for refining my experimental method will be highly appreciated.

Run some of the qPCR results on a gel or have a look at the melt curves - you might well have more than one product being generated. Your std curve should have a little over 3 cycles between each point (copy number doubles each cycle so 3 cycles is 8x more copies to reach Ct) and the replicates should be quite tight. How long is the PCR product? It shouldn't need extension if it is <100 bp.

@bob1 I did, and you are absolutely right. Indeed, I have two zones on the curve amplicon. How do you know that? Wow, I am just surprised. Does it mean that my primers were not extended correctly? The primer sizes are 194 and 162 bp.

@KGee It was a suspicion (MikeyC noted it too), experience is a great tool... Your primers are 194 bp and 162 bp each? Or are those the amplicon sizes? Primers should only need to be about 18-25 bases for a highly specific reaction. The manual for the Kapa pol says extension of products less than 400 bp should need 1 second.

@bob1 Oh, I didn't get that (MikeyC) mentioned double zones. Obviously, you are an expert user. :) Do you recommend adjusting step 3 from 3 to 1? Will this extension fix the issue of the double zones? The f primer is 194 (22 bases), the R one is 162bp (20 bases), and the amplicon size is about 150bp. Thank you again!

Ah, I see - the primers are called "194" and "162". The sizes 20 and 22 are fine. You should go and optimize the binding of the primers in the Kapa mix; it might well be different to in another system.  A 3-step is fine. The Kapa manual says  For 3-step cycling protocols, anneal at optimal annealing temperature
for 20 sec followed by the minimum time required for data acquisition
at 72ºC according to instrument guidelines)  So you should acquire only at extension and for the minimum capable on your instrument. You'd need to check the instrument manual for this information.

@bob1 Oh, I see. Thank you once again for the response. Let's see. Then I hope to "get rid" of double zones this time  :P!!!