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Which modern methods of antibiotic production are there?
For my seminar paper in the field of biology, I have to collect modern methods of antibiotic production. The topic is a little overwhelming, but the work should also be quite extensive (approx. 18 pages).
I've already done some research, but most of the results don't help me. I would be very grateful for a few web links and keywords that I could research for in order to break down the topic.
I will visit the university library in the next few days. It would help me a lot if someone could recommend a few good books references.

Are you interested in antibiotic production (so via chemical synthesis, metabolic engineering of microrganisms or fungi, etc..) or antibiotic R&D (compounds library screening, drug design, protein-protein interactions..) ?

I remember being told that making antibiotics against gram negative bacteria is a little bit more challenging than gram positive due to outer membrane differences. As such I'd recommend reading this paper which is great (Predictive compound accumulation rules yield a broad-spectrum antibiotic): https://www.nature.com/articles/nature22308 They dissect the efficacy of antibiotics for gram negative bacteria by outlining certain requirements, such as rigidity, globularity, presence of amine groups, and amphiphilicity. Also molecular weight, and polarity play a role.

For Penicillin production try from page 165 of:
Industrial Microbiology: An Introduction
by
Michael J. Waites
Neil L. Morgan
John S. Rockey
Gary Higton
There is also a table in that chapter listing the other antibiotics and which organisms are used to produce the basic form.
I found it very readable.


text:
AMPK, PAN-AMPK, western blot
What does it mean by adding PAN before the AMPK. I am trying to do a western blot assay on AMPK and I am confused by PAN-AMPK, AMPK. does it mean pancreases? Is pan-ampk the total ampk?

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Can you please give some more background of your research?

Adenosine monophosphate activated protein kinase or AMPK is not a single protein, but is a trimeric enzyme composed of $\alpha$, $\beta$, and $\gamma$ subunits. There are 2 alpha genes, 2 beta genes, and 3 gamma genes, each coding for closely-related but not identical isoforms. In addition, like most protein kinases (and many other proteins), it is regulated by the phosphorylation and dephosphorylation of some of its residues, most notably Thr172, but at numerous other locations as well.
A "pan-AMPK" antibody will bind all of the AMPK isoforms independently of phosphorylation or other post-translational modification (PTM) state. There are also isoform-specific total AMPK antibodies that just bind the $\alpha2$ subunit, or all 3 $\gamma$ subunits, for example. Finally, there are phospho-specific antibodies to various residues that only bind when the protein is phosphorylated at that site.
Both the pan and isoform-specific antibodies are known as "total" antibodies, as they bind independently of PTMs and will show the total amounts of their target(s) on a Western blot, for example.

Source:
I used to be a Product Scientist for a company that makes antibodies, PTM-specific and total, to AMPK and many other targets.

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How long might a tick be on a body before biting?
I understand that a tick must be attached for roughly 24+ hours before presenting a danger of transmitting Lyme Disease. However, I am wondering how long a tick might be on my body before actually attaching itself to me.

Anecdotal: I have found ticks that have not fed on me hours after I've left the forest and changed clothes (and avoided areas I might have exposed to wayward ticks). This has certainly been the exception out of the 1000s of ticks I've had on me, but in my experience is not improbable. CDC is often a good source for tick info, by the way.

They wander the body for 30 minutes to an hour before inserting their feeding parts into the skin. Source.
It entirely depends what material you are wearing, i.e. fluffy wool is difficult for them to climb on. They can fall off somewhere in the home and bite another day.
2mm nymphs climb on human skin at about 10cm per minute, so it could find the back of the knee in about 5 minutes and search for a bite site. tick attachment site distribution is: 9% head-neck, 5% arm,
24% stomach-groin, 7% back, 18% chest-shoulder, 25% leg-foot, and 12% hip.

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What is the significance of running an uncut plasmid on electrophoresis gel?
What is the significance of running an uncut plasmid on electrophoresis gel?
In this case we are talking about inserting a gene into plasmid, which then goes under PCR and then electrophoresis.

So you clone you DNA, make a PCR and then run the plasmid on a gel? This doesn`t make sense, can you post your complete protocol?

It is used to determine whether the unused/excess amount of plasmid has contaminated the  final solution of the desired recombinant DNA  or not.
Electrophoresis is performed on the "Blank"(uncut) plasmid to define its bands.If the electrophoresis result contains the bands of both "blank" and recombinant DNA , it shows that your solution is not 100% pure.


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Transformation and PCR in molecular cloning
After obtaining the recombinant DNA, it is common to transform into E. coli to screen for recombinant DNA and amplify it. But I would like to ask can we amplify it using PCR?
I think there will be 3 DNA molecules after restriction-ligation:

recombinant DNA 2. self-ligated plasmid 3. unligated fragment containing the gene of interest (GOI)

To amplify recombinant plasmid only, can we design primers that span through the insert fragment and the vector, which can only pair well with the recombinant plasmid, but other types?
However, I don't know whether this restriction in primer design will affect GC%. So I am thinking whether we can do phosphatase treatment to prevent self-ligation, then add exonuclease to digest all linear DNA i.e. only retain recombinant plasmid eventually, so the restriction can be overcome (since we can design primer on suitable location on recombinant plasmid now), may I ask is this valid?
After PCR, the recombinant plasmid is linearized (2 blunt ends created by forward and reverse primers if I understand correctly?), which becomes less stable since it can be degraded by exonuclease. Therefore, can we add ligase to circularize the plasmid? Can ligases ligate 2 blunt ends?
I don't know whether this method can be done but I am thinking if we just want the amplify the DNA (don't need the protein product) after generating recombinant plasmids, won't this be more efficient than bacterial transformation?
Thank you and please correct if I made any mistakes.

why would someone go through the hassle of cloning a fragment into a plasmid if you have no intention to use the plasmid for anything?

I’m voting to close this question because it is unclear, seems to contain multiple questions, and shows no evidence of the expected prior research. Please note that each of those is sufficient reason for closure on this site. Please see the [help] starting with [ask] for details. It also appears to be based on a misunderstanding of how and why molecular cloning is done.

I'm not at all sure that you recognize the process here. Generally the bacteria are used for amplification of the plasmid to levels at which you would use it. Doing this by PCR is technically possible, but much much more expensive, has a higher error rate (due to error prone DNA polymerases), and requires extra steps. You also won't get as much DNA out as you would from a bacterial culture. Culture is also scalable - you want more DNA, just grow more bacteria. Once you have a plasmid transformed into a bacterial strain, you can store stocks of this at -80 in glycerol form. You can then scrape a small bit, grow overnight and perform a <2h procedure the next day and end up with several micrograms of very clean DNA.
I highly recommend you read a good cloning protocol, such as those found in Sambrook et al., Molecular Cloning: a laboratory manual or Current protocols. Cloning has been around for >50 years now, I think the process is more or less optimized.
The usual process of generating a plasmid goes like this:

Generate insert (often by PCR, but maybe from another plasmid or via a library generation)
Digest insert with appropriate restriction enzymes
Digest backbone with the same enzymes
Ligate backbone and insert
Transform into bacteria
Pick colonies
Screen colonies for insert (See below)
Grow up colonies with successful clones
Prepare plasmid DNA from successful clones
Use that plasmid in experiments.

The bacteria will generally digest the linear DNA through production of exogenous DNases, and linear DNA often doesn't transform well, so your exo-nuclease step is unnecessary.
Usually when you are screening colonies you use primers specific for the insert, or, if wanting to determine if the insert has been inserted in a specific orientation, then one primer on the backbone and another on the insert. Only amplification from both primers producing a band of the correct size is a positive insert. In this case it is possible to produce primers that only recognize those plasmids that have an insert.
There are a few cases where you might generate primers that span the whole insert - sequencing of the whole insert might be one. Plasmids often have these sorts of sites already incorporated. If you see something labeled M13 or BGH, these are common sites for sequencing primers. Another common usage is if you want a range of inserts (e.g library preparation), and you don't know how big the inserts might be, then you can use the insert spanning primers to identify plasmids which have self-ligated (no insert) as these will only produce very small products on a gel. It is not possible to make insert spanning primers that only recognize the recombinants. No need for ligation or exonucleases.
Another possible scenario is where you want to change a base (or more than 1) in an already constructed plasmid. This is called Site Directed Mutagenesis. In this case you do want the PCR to go all the way around the plasmid, and parental DNA (from the plasmid) is damaged by digestion with the methylation specific restriction enzyme DpnI (PCR products are not methylated, but bacterially grown usually are), and the nicked circular product repaired by the bacteria after transformation.
Primers will not affect the GC content of your product. If you don't like the GC content of your primers, move them to a more amenable location.
It used to be that everyone did an alkaline phosphatase (APs) treatment of the digested DNA to prevent self-ligation. In practice, APs are very hard to get rid of or inactivate and tend to remove the necessary phosphate from your insert when you mix the products together at ligation. This leads to inhibition of the ligation and lowers your chance of success.


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Procedure of diagonal electrophoresis
I am learning protein sequencing and it seems that diagonal electrophoresis is a common method in the identification of disulphide bonds (S-S) exist between polypeptides in a protein.
Questions:

After horizontal electrophoresis, we need to cleave the S-S bond using performic acid. According to Wiki,

Its strong oxidizing properties are used for cleaving disulfide bonds in protein mapping.

However, from what I have learnt in SDS-PAGE, 2-mercaptoethanol OR dithiothreitol are commonly used for cleaving S-S bonds, may I ask why do we need to use formic acid specifically here?

the first electrophoresis is done horizontally and the second is done vertically (bottom to top). May I ask whether we can do it vertically then horizontally instead? It seems to me that it should be OK since there doesn't exist any disruption to the proteins but I would like to confirm.

Thank you.

You probably don’t need to oxidize the disulfide bonds rather than reduce them. Performic acid has the advantage that the oxidation products can’t spontaneously reform a disulfide bond whereas the reduction products from a thiolate reductant can. This may not be a significant issue in the actual application. Oxidation by performic acid also causes a significant mass change for the cysteine residues which can be useful for downstream mass spec. These oxidation products are also negatively charged which can increase mobility in PAGE and possibly aid separation.

@canadianer thanks for pointing out my mistake :) So can I say: performic acid oxidize the sulfur in cysteine residue, forming cystenic acid i.e. disulphide bond is cleaved. Moreover, this is the cause of 'significant mass change' (-SH --> -SOOH), which can be utilized by MS for easy determination of S-S bond (where B-ME and DTT cannot achieve this)? Thanks

ref: https://www.researchgate.net/profile/Tanea-Reed/publication/51828850/figure/fig11/AS:668669944279047@1536434746552/Cysteine-oxidation-at-neutral-pH-Cysteine-plays-an-important-role-in-the-regulation-of.png

This article states that complete oxidation of the cystine to cysteic acid occurs (SO3-), which is a molecular mass change of 48 u. With BME or DTT reduction, you are left with a thiol or thiolate.

Now I understand thank you very much

You’re welcome.

You probably don’t need to oxidize the disulfide bonds rather than reduce them. Performic acid has some potential advantages:

The oxidation products can’t spontaneously reform a disulfide bond whereas the reduction products from a thiolate reductant can.
Oxidation by performic acid causes a significant mass change for the cysteine residues which can be useful for downstream mass spec. This article states that complete oxidation of the cystine to cysteic acid occurs (-SO3-), which is a molecular mass change of 48 u. With BME or DTT reduction, you are left with a thiol or thiolate.
These oxidation products are also negatively charged which can increase mobility in PAGE and possibly aid separation.


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Ensuring all individuals are genetically identical before experiments
I am a beginner in experiment design so please correct me if I made any mistakes. If we want to study the behavioral changes after a particular mutation of a gene, it seems to be a must for all individuals to be genetically identical before we induce the mutation (otherwise those changes may not be caused by the mutation, but other genetic variants)
For example, if we are using fruit flies for genetic study, we need to enclose them (with food and air) such that they can only mate over many generations in that container.
Question: After many generations, supposedly a very large number offspring which has very similar genetic makeup can be generated ready for mutagenesis. However, may I ask how to test whether they are 100% genetically identical? I have thought of gene sequencing, but is highly impractical since there is a very large population involved.

For fruit flies one usually wants pure lineages for specific alleles, corresponding to distinct phenotypes. One cannot arrive to such a state by merely enclosing the flies for many generations - in this case one will quickly end up in the situation of Hardy-Weinberg equilibrium, where all the alleles are present in constant proportions, not changing with time. Of course, in a finite population one expects that eventually one allele fixes  itself, but that may take a rather long time in absence of selection. Thus, in practice one separates the desired phenotypes in every generation, and and makes them breed among themselves. In fact, this is pretty much what Mendel did with peas to obtain pure lineages - it is worth reviewing this chapter.
Now, it is always risk that the population is not 100% pure, just as there is always a possibility that other genes contribute to the trait of interest. Here is where the (bio)statistical analysis comes in: testing for significance of the effect and filtering out random effects.
Update
Just to provide a quick calculation: suppose that we have alleles A and a, with A being the dominant one. In every generation we remove the undesired genotype aa. Initially we have the genotype frequencies of  AA,Aa,aa given by $P, H, Q=0$,
where $P+H=1$. The allele frequencies are $$p=P+\frac{H}{2},q=\frac{H}{2}$$
After crossing we obtain the allele frequencies $p^2, 2pq,q^2$, which after removing the aa genotype give:
$$
P'=\frac{p^2}{p^2+2pq}=\frac{p}{p+2q}, H'=\frac{2pq}{p^2+2pq}=\frac{2q}{p+2q}
$$
The new allele frequencies are
$$p'=\frac{p+q}{p+2q}=\frac{1}{1+q}, q'=\frac{q}{p+2}=\frac{q}{1+Q},$$
where I used the fact that $p+q=1$.
More generally, the frequency of the unwanted allele in generation $t+1$, in terms of the frequencies in the previous generation, is given by
$$
q_{t+1}=\frac{q_t}{1+q_t}
$$
The solution to this difference equation is
$$
q_t=\frac{q_0}{1+tq_0}.
$$
Now, if we want the frequency of the unwanted allele to be below teh desired threshold $\alpha$, we must solve inequality $q_t<\alpha$, obtaining
$$
t>\frac{q_0}{\alpha}-1
$$
generations. A good choice of $\alpha$ is $1/N$, where $N$ is the number of individuals in the population (than after $Nq_0-1$ generations we are likely to have no more than one individual carrying the minority allele).
Remark
A similar calculation can be found in connection with strong selection, under conditions of complete dominance: if $s=1$, the aa genotype is completely eliminated at each generation. See, e.g., Introduction to Quantitative Genetics by Douglas Falconer.

People have already done the inbreeding over sufficient generations for plenty of fast-breeding model organisms, and you just buy your initial stock from a company, with the understanding that some small amount of genetic drift may occur over time.

Normally the companies/organizations that supply inbred animal strains take precautions to minimize drift, such as keeping a large stock of frozen founders and periodically refreshing their working populations from those.  They also monitor their working populations' genomes, these days presumably by whole-genome sequencing.  Details vary by species and organization.


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Electrochemical and concentration gradient confusion
I am learning membrane transport and learnt that there are 2 gradients that determine passive and active transport.

If the concentration of a cation is higher in the cytoplasm side (hence ions should flow out of the cell passively), but there is higher electrochemical potential in the extracellular side (hence ions should flow into the cell), which side should that ion flow if there is only passive transport?
I know there is Nernst equation for electrochemical potential, but I would like to ask whether there are equations for calculating potential caused by the concentration gradient? Since my thought is by comparing which has the higher potential difference, and deduce which gradient is dominant, eventually deduce the direction of the net passive flow of that ion.
Thank you

All you need is the Nernst equation and the voltage across the membrane.
The answer you get from Nernst is the equilibrium potential for the ion of interest. This is the potential at which the concentration and electrical gradients are exactly equal for that ion. If the voltage across a membrane is at the Nerst potential there is no net passive flow of the ion.
If the voltage is different then the Nernst potential, then the ion moves in the direction that would tend to move the voltage towards the Nernst potential. If the Nernst potential is -90 mV, the membrane potential is -60 mV, and you have a positive charged ion, the ion will tend to move out of the cell because this is what would move it closer to -90 mV.
You can't separate the chemical and electrical gradients and sum them up or anything, the specific way to combine them is given by the Nernst equation. The Nernst equation is not for calculating the electrical potential separately of concentration gradient, it's for calculating the combined result of electrical and chemical gradients. The way to know which way an ion flows is the way I've described it, not by determining which is "dominant".


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Washing buffers for protein identification?
I am currently learning protein identification techniques and come across ELISA and Western Blot. In these methods, a washing buffer is required to wash out the antibodies that are not bound to the plate (the specific antigens).
Questions:

I understand that detergent (E.g. Tween-20 (T)) is usually added since it can wash away the non-specific binding of antibodies. (ref) However, from what I have known, we are also adding blocking buffer to block the unoccupied sites on the membrane, which also prevents non-specific binding of antibodies (ref). I would like to ask if this is true, why do we need to add Tween-20 to the washing buffer?

I have heard PBST and TBST are 2 common washing buffers, where TBST seems to be much better. From ThermoFisher:

Determining the proper blocking buffer can help to increase the system’s signal-to-noise ratio. Occasionally, when switching from one substrate to another, the blocking buffer may need to be changed in order to avoid problems with diminished signal or increased background. For example, with applications using an alkaline phosphatase (AP) conjugate, a blocking buffer in Tris-buffered saline (TBS) should be selected because phosphate-buffered saline (PBS) interferes with AP activity.

This seems to me that TBST is always the preferred option (also ref). Then I would like to ask in what specific situation, that PBST is preferred than TBST, otherwise, it will cause a major problem to the result e.g. signal/noise ratio?
Thank you.

All the unoccupied sites on the membrane are saturated in the blocking step after the transfer of the protein gel to the membrane. You can prepare the antibody solution in pure buffer, but you should expect a slightly higher background (this of course depends also on the antibody used), since there still may be some binding possible. When you have a much higher amount of blocking agent in this solution it is much more unlikely that the antibody is able to bind non-specifically, since these sites are occupied already.
Some low affinity binding occurs between the antibody and other proteins which you will not be able to wash away without a detergent.

In my experience it doesn't really matter for most cases which buffer you use as long as you do it consistently. There are of course a few exceptions:

When working with phospho-proteins PBS can give a much higher background - but it can also work. It needs to be tested and optimized, but it is probably easier with TBS.
When using alkaline phosphatase in your detection step, you cannot use PBS since free phosphate inhibits the enzyme.


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How to identify plasma cells that only produce monoclonal antibodies?
I am studying the procedures of forming hybridoma cells for generating a large number of monoclonal antibodies.
Before the procedure of fusion (with multiple myeloma cells) happens, I would like to ask how to ensure all the plasma cells in the cell extract only contains those which produce monoclonal antibodies only but not polyclonal antibodies?
I have searched in Wiki, and it says

identifying antigen-specific plasma/plasmablast cells (ASPC) that produce antibodies specific to an antigen of interest

I would like to ask what methods are available for us to do this identification?
Thank you.

You don't do this before the fusion, you do it afterwards. First you make the hybridoma cells, which are then seeded as single cells into multiwell plates and cultivated to get enough cells for testing and further culture. This way you get only one antibody type produced per well (which is coming from a single clone, hence it is monoclonal) which then can be isolated and tested for antigen specificity.
For a schematic see the image from this publication:

Oh thanks, so using my words (to clarify my understanding), can I say: putting individual (different types of) hybridoma plasma cells into different wells, so 1 type of cell in 1 plate --> 1 specific antibody (hence prevent polyclonal Ab formed since there are no signalling of 1 cell to multiple plasma cell lines to produce them)?

And may I ask is the purpose of screening after fusion is to make all of them immortal before culturing (which takes a relatively long time)?

@Questions Your wording "...there are no signalling of 1 cell to multiple plasma cell lines to produce them)? " is confusing to me - what do you mean by "signaling"?

@Questions The purpose of screening is to identify which clonal line is making a desired antibody - remember each clonal line (each well) makes a different antibody since it is descended from a different B cell. The immortalization has already happened from the fusion with immortal myeloma cells.

@Armand Thanks for clarifying, now it is very clear to me :) and sorry that I have used the wrong phrase since I just want to confirm that in individual wells, there are no other plasma cells present to interrupt.

@Questions Sorry for the delay, I had other stuff to do. You do the screening after the generation of the immortal cells because you can expand these (theoretically at least) forever. And generation of this cells is nothing which is 100% effective. This way you screen cells which are immortal, can be used for antibody production. If you do it the other way, you cannot be sure to get immortal cells from your screened cells.


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Bug identification request
Can anyone please offer a suggestion of what the insect below is?  It is about 3mm in length, and I saw it on my kitchen counter.  I live in an apartment, 9th floor of a highrise.  It is January 5 (winter), 2021, in Ottawa (-3C outside).
I suspect that is might not be relevant, but last month, I saw what might be Dermestid lavae.  The beetle below, however, doesn't look like a Dermestid beetle
Thanks.


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Why is neuronal firing rate proportional to axonal diameter?
I know that a larger axonal diameter results in faster speed of action potentials due to reduced resistance. However this doesn't explain why the firing rate of neurons is proportional to neuronal diameter. Is it because higher spike rates require larger capacitance (since each spike charges the neuron) meaning a larger surface area and diameter of neuron is needed?

Can you quote a source for firing rate being proportional to diameter?

Principles of Neural Design (Simon and Laughlin), chapter 3 page 54. Also https://pubmed.ncbi.nlm.nih.gov/19525561/ (Balasubramanian, Sterling 2009)

Also a short quote from each and edit into your question please (comments are meant to be temporary).


text:
Is it plausible that strict lockdowns made it more likely for the new variant of COVID to have emerged?
My idea is that strict lockdowns put greater evolutionary pressure on the coronavirus by restricting oppurtunities to be transmitted, meaning that a faster-spreading variant had much less competition.
Is this at all plausible? Was it just as likely for a faster-spreading variant to become the predominant one without lockdowns?

It's actually a complicated trade-off between the total amount of time that the virus has time to mutate and number of hosts it can mutate in. You've basically selected the wrong answer. aliential's answer links to a paper that does this sort of (non-trivial) analysis. I'll also note there that it's much harder to quantify the chance that rare "human lab" events can occur, which are thought to be at the origin of the latest UK variant of concern. https://virological.org/t/preliminary-genomic-characterisation-of-an-emergent-sars-cov-2-lineage-in-the-uk-defined-by-a-novel-set-of-spike-mutations

Okay, I guess that I am not going to select an answer, as I am not really qualified to judge. That answer made sense to me at the time.

@kCODINGeroo Why do you ask a question if you aren't qualified to accept an answer? Only you as the original poster can accept an answer.

While I get your intuition, the hypothesis seems implausible

Emerge of new CoV variants should be considered proportional to the
current spread of the virus. Each virus has a certain mutation rate that surely does not depend on lock-downs.
It seems implausible to interpret
lock-downs as physical limitations to viral spread, that could potentially be
overcome by any mutation.
Even if CoV magically learned to
teleport to different households, such an ability would always represent immense advantages; an advantage that does not need lock-downs to be effective
Lastly, the differential viral competition is neglectable so far; we are at 6 of 100 thousand infected persons. So basically a 0% chance that a virus would infect a person who has acquired an immunity due to a previous CoV infection. In your model this person, who is immune, further would need to NOT be immune because quarantine prevented the (previous) infection.

This is more plausible (second point below) and less plausible (third point below).

Selective advantage The emergence of a new strain of a virus relates to the selective advantage (Gordo 2009). This selective advantage might be that the reproduction rate is relatively higher.

Reproduction rate The selective advantage (in terms of relative growth rate) is based on the relative growth rate of the virus. Because the growth rate for viruses is not directly proportional to the reproduction rate (Wallinga 2009), this means that better selective advantage is not exactly the same as better reproduction rate (and that is just the selective advantage due to reproduction rate). Instead, it is more like related to the reproduction rate minus one. The relative (initial) growth rate of the mutated virus (made dimensionless by multiplying with generation time) gives the relative growth factor  $$\text{growthrate} \times \text{generation time}  = \overbrace{s}^{\text{selective advantage}} = R_{mutant}- R_{other}$$ If you have some mutation that gives an improved effective reproduction factor $R_{mutant}/R_{other} = c$ then the difference $R_{mutant}- R_{other}$ will be larger for a larger $R_{other}$. This is not the case with a lockdown ($R$ will be typically lower) and in a situation with a specific ratio $R_{mutant}/R_{other} = c$ the relative growth of the mutant will be smaller if $R_{other}$ is lower

What makes reproduction rate higher? There is a small catch with this second point (from which you could conclude that growth of new strains is higher without lockdowns). Another non-linearity is that the reproduction rate is not scaling linearly with the infectivity of the virus.
For instance Ferrari et.al. (2006) model the probability of infection as an exponential function of the rate of transmission

The susceptible nodes become infected in the next time-step with binomial probability 1−exp(−βI), where β is the rate of transmission across an edge, and I is the number of infected nodes to which the individual is connected.

The infections are a function of time and proximity (and also the susceptibility and infectiousness of people), and this is not homogeneously distributed. This makes that a simple reduction of 'infectivity' by half is not at the same time also making the effective reproduction rate become halved.
You could see the reproduction number being buffered in some sense. There is an excess/overflow of transmission in some parts of the network. When you are sick, then most likely your family members are gonna get sick as well. When the virus becomes half less transmissible, then it is not like only half your family members are gonna get sick. Reducing the transmissibility might have an effect for short-term contacts (which are important pathways as well), but it is of less influence for intensive contacts.
This buffering makes that the transmissibility of the virus (the reproduction rate) is not so much controlled by the changes in the properties of the virus. The transmission is more controlled by the human traffic and contact network (and the fluctuations occurring within this network/traffic). If an average person sees, on average, only 3 people within the infectious period, then the basic reproduction number will be at most 3 and it can't suddenly make a big jump. You can not infect more than 3 people if you do not see more than 3 people, so the virus can become more transmissible but it won't influence the reproduction rate 'as much' (unless there is an entirely different mode of transport). (I am putting stress on 'as much' because there will be some influence, just like adding acid to a buffer will not have zero effect, but it just won't be the same)
Any growth of small mutations in new strains is (initially) more likely to be occurring randomly. Viruses mutate a lot, and there will a lot of probability that there is a lucky sample (many others will be unlucky) that might accidentally reach some area with a fast growth rate and with little competition (isolation from competition is a good, but also likely, route for a non-competitive mutation to grow in number). This randomness becomes larger when the total numbers are smaller. If a new strain hits a (relatively) clean area then it will spread out without any/much competition and it can become big (especially if it is an area without much control) and spread over a large area unnoticed.
This isolation might be more likely in a scenario with strict lockdowns, which makes the spread of the virus more chaotic (it's like rolling a dice a few times, you'll be more likely to get the same number with a high frequency in comparison to rolling the dice often; the lockdowns make the spread dependent on fewer dice rolls and give more chances to some mutation to slip through, reach some prosperous less lock downed area, and become more dominant)

So, in general there is no clear influence from a lockdown. In general the emergence of new variants relates to Fisher's fundamental theorem of natural selection

"The rate of increase in fitness of any organism at any time is equal to its genetic variance in fitness at that time."

The second point above (lower fitness/growth-rate results in lower natural selection) relates to the fact that it we reduce the fitness with a factor $c<1$ by a lockdown, than the variance of the fitness changes by a factor $c^2$. The change of the fitness will scale with $c^2$ and the relative change of the fitness will change with $c$.
The third point above relates to a breaking of the assumptions (there's no homogeneous spread of the virus). Breakdowns can create relatively separate/isolated regions and the spread may not be via a Malthusian growth model but instead more chaotic (with random branches like a thunder bolt). This makes it not unthinkable that a variant emerges quickly by a random effect and without much difference in selective advantage.
Some references

Gordo, Isabel, et al. "Genetic diversity in the SIR model of pathogen evolution." PloS one 4.3 (2009): e4876.

Wallinga, Jacco, and Marc Lipsitch. "How generation intervals shape the relationship between growth rates and reproductive numbers." Proceedings of the Royal Society B: Biological Sciences 274.1609 (2007): 599-604.

Ferrari, Matthew J., et al. "Network frailty and the geometry of herd immunity." Proceedings of the Royal Society B: Biological Sciences 273.1602 (2006): 2743-2748.

Current Biology 30, R841–R870, August 3, 2020 R857, does support some of your theory.
The paper summarizes the scientific basis of their statements:

Even though the adaptive significance of genetic variants remains to
be established, we can use evolutionary theory to gain insights about
how natural selection might act on disease characteristics.

The mutation rate is related to the population size which has stayed nearly as high this winter as it was with dense crowds previously, regardless of distancing, so the following statement is all the more likely,

some mutations may become more strongly selected in the presence of
social distancing if they allow for viral transmission despite the
intervention (for example, mutations affecting aerosolization or
persistence in the environment).

i.e. physical distancing will select for better breach of distancing, and quarantineing will select for higher transmission during the asymptomatic days prior to fever.
The transmission methods aren't very well know, i.e. asymptomatic transission, transmission by children, peak transmission before symptoms appear, rates of contact Vs airborn transmission.
So, you are right, distancing encourages mutants that reproduce and transmit despite distancing tactics.

Do lockdowns help new mutations emerge? - No.
Mutations appear at an approximately constant rate in every genome replucation event. In this sense, the lockdowns reduced the overall number of mutations, by reducing the number of infections and new replucation events.
Could lockdowns help new mutations become prevalent? - Possibly. Whether a new mutation survives and becomes widespread depends on its fitness, vis-à-vis the adverse environment in which the virus exists. The more contagious viral strain has a selective advantage over the other strains. The speed with which such a strain pushes out the other strains out of the existence, i.e., the time that it needs to fix in the population, depends on the effective population size. In this respect, lockdown resulted in reducing the effective population size, which could have resulted in faster fixation of the more contagious strain. (It is important to keep in mind that the lockdown wasn't waterproof - the virus was still spreading, although at a lower pace.)
In other words, the lockdown could have acted as an evolutionary bottleneck, reducing the viral diversity at the expense of the less fit strains.
Has this really happened? - No. We are not dealing with this situation, but rather with a viral strain that has emerged only recently, which is certainly far from having fixed in the population. One cannot even be sure that it has a selective advantage - it is more contagious, but may have other, yet unknown, shortcomings.
If the new strain does have selective advantage, the future lockdowns might "help" it to become the dominant strain. This shows that lockdowns are not sufficient to eradicate a virus  - as opposed to completely quarantining/isolating whole groups of population, as was done in China or during the Ebola outbreaks. Eradicating the virus however never was the point of the lockdowns: they were implemented to slow down its spread, so that it does not overwhelm healthcare systems, so that effective testing procedures could be put in place, and to allow time for developing vaccines.

Your first answer on "Do lockdowns help new mutations to emerge" assumes that the spread can be stopped by the NPIs. If, however, the spread can't be stopped, but only limited, it's my understanding that it will help new mutations to emerge. Furthermore, NPIs apart from complete isolation, can introduce strains that are more transmissible, as they're the only ones that will survive

@Highmastdon I didn't make such a strong claim - I meant that the lockdowns reduce the basic reproduction rate, which means less infectioned eprsons and consequently less replication events for virus... for a period of time - if no other measures are taken, the eventual  number of the reproduction events may be just as high, although the effective population size still remains lower.

You are the victim of the "post hoc ergo propter hoc" fallacy: just because event B follows event A doesn't mean that B is caused by A. The probability of a mutation to occur depends on the number of virions out there multiplied with some constant that models the faithfulness of the virus reproduction machinery. The lock-down was instituted because the number of infected persons - and hence virions - was high. Thus, both the lock-down and the occurrence of mutations share the same cause, one of the possible reasons for nonsense correlations.

I don't think the question makes any claims (and thus commits a fallacy) - it is more an inquiry about a possible relation between two events. Also, emergence of a new strain depends not only on the availability of mutations, but also on the evolutionary pressures, the random drift, the population structure - all of which were affected by a lockdown.

The OP - whether formulated as question or as statement -  contains a hypothesis. This hypothesis is based on a nonsense correlation. And the probability of a mutation arising depends on the factors I mentioned; the factors you named determine whether this mutation can spread.

the OP is asking about the emergence and spread of the new strsin, not just about a mutation. In fact, they even do not mention mutations.


text:
Difference between diffusion and osmosis
Osmosis in plants so called diffusion too. Need to understand the difference between osmosis and diffusion Because this both process transport molecules from region of higher concentration to region of lower concentration.

See in diffusion co2 o2 gets transported from high concentration to low concentration through semi permeable membrane and And in osomosis also water gets transported from high concentration to low concentration through semi permeable membrane

Does this answer your question? When does DIFFUSION occur and not OSMOSIS, and the otherwise?

Because this both process transport molecules from region of higher concentration to region of lower concentration.

This is not strictly true. Molecules don't move in the same direction, rather the concentration moves towards equilibrium (higher to lower)
In Osmosis, the first characteristic is a semi permeable membrane. (More on such membranes in College level biology). Secondly, it is the solvent molecules that move, so that concentration is made equal on both sides of the membrane.

This is the most important feature in osmosis. Solvent molecules move, from a region of low concentration to higher concentration. When solvent molecules increase in a region of higher concentration (right side in the image), the concentration comes down; while that in the region of lower concentration (left side in the image), increases.
On the other hand, diffusion is the movement of the solute molecules. If you were to spray a perfume in one corner of a room, the smell would then be felt everywhere else. This is because the perfume molecules are diffusing through the room.

In a different example, suppose you have a box with a partition, full of gas. One side of the partition has the gas at high pressure and the other side has a low pressure. When you remove the partition, molecules of the gas will diffuse from the side with higher concentration to the side with lower concentration.
In summary, the difference between osmosis and diffusion can be found in identifying WHICH molecules move.


text:
Species Identification: Waterborne worm/larva, California, USA (and is it safe)
Can you help ID this species? The photos are low quality, so I'll do my best to describe them.
I found them after I left a cup of dechlorinated, freshwater aquarium water outside with a plant in it, hoping the plant would grow better in sunlight. I do not think they came from the aquarium, but I might as well add it.
I left this cup on my second floor balcony. I don't know if this might narrow down the search, but whatever insect it is is somehow present on my roof (flying or climbing, I don't know).
They have a black head, are visibly segmented inside, but their outside is smooth, with no ridges. They are about 3-5mm long (I think based on age). I put them on a bit of cardboard paper to take the photos, and as the paper dried it appears that they need water or at least a moist surface to survive.


text:
What does "culture" mean here?

India becomes first country to culture U.K. variant strain.

Source
What does "culture" mean here?

Are you asking about the exact culture conditions? (The general meaning of "culture" was given in the source: "Culture is the process by which cells are grown under controlled conditions, generally outside their natural environment.")

Since viruses cannot reproduce outside of a cellular environment, they are cultured by infecting a living culture of host cells. In this case, it means human cells are cultured in a sterile dish and infected with a specimen of SARS-COV-2 virus, most likely collected from a patient during the early stages of infection. After infection of the cultured cells is confirmed, the new virus particles are collected from the culture media and put into storage.

Why does this need citations? This is just knowledge picked up from working in a virology/cell biology lab.


text:
Review paper on gene expression regulatory mechanisms
I'm a junior grad student in bioinformatics with CS background. I am involved in a project around discovering gene regulatory networks in some data, which got me curious about the gene expression regulatory mechanisms in eukaryotes. I found this page on Nature, which looks super helpful:
https://www.nature.com/collections/pfdsgqgzck
But it does have 27 review papers listed there. Are there any review papers you could suggest that sums up/introduces different regulatory mechanisms and regulatory factors (CTCF, chromatin accessibility, TFs, enhancers, promoters, lncRNAs etc) in a more global overview?
Thanks!

That page looks to be a promotional effort for their review articles, organized by recency and selected by some keyword like 'regulation'.


text:
How can I convert plate reader measurements of Pichia pastoris OD to cells per ml?
I am performing experiments with the yeast Pichia pastoris where it will be beneficial to calculate product yield per cell. I plan to use a plate reader to measure OD600 of my cultures and then somehow relate this to cell number.
The Pichia pastoris expression manual from ThermoFisher says that 1 OD600 is equivalent to 5x10^7 cells/ml, and Weis et al (2004) have a similar number of 1.5x10^8 cells per 1 OD.
However, there are now calibration protocols (Beal et al 2020) and packages (Fedorec et al 2020) for using absorbent beads to convert OD measurements to cell density in E.coli. These rely on the beads scattering the light in a way that is equivalent to cells, so that one can compare measurements of OD with a standard curve of known amounts of beads to estimate cell number. Pichia is about 5µm in size size, compared to E. coli which is about 1-2µm, therefore I do not believe that using the same protocol will work for me.
Is it necessary for me to develop a new protocol, or can I just convert by multiplying OD by the numbers given above? If it is necessary, how can I develop such a protocol? Can I just use the E. coli protocol that I linked with different size beads?
Refs:

Weis, Roland, Ruud Luiten, Wolfgang Skranc, Helmut Schwab, Marcel Wubbolts, and Anton Glieder. ‘Reliable High-Throughput Screening with Pichia Pastoris by Limiting Yeast Cell Death Phenomena’. FEMS Yeast Research 5, no. 2 (1 November 2004): 179–89. https://doi.org/10.1016/j.femsyr.2004.06.016.
Jacob Beal, Traci Haddock-Angelli, Markus Gershater, Vishal Sanchania, Russell Buckley-Taylor, Geoff Baldwin, Natalie Farny, Richard Tennant, Paul Rutten 2020. Calibration Protocol - Plate Reader Abs600 (OD) Calibration with Microsphere Particles. protocols.io
https://dx.doi.org/10.17504/protocols.io.bgy6jxze
Fedorec, Alex J. H., Clare M. Robinson, Ke Yan Wen, and Chris P. Barnes. ‘FlopR: An Open Source Software Package for Calibration and Normalization of Plate Reader and Flow Cytometry Data’. ACS Synthetic Biology, 27 August 2020. https://doi.org/10.1021/acssynbio.0c00296.

Is there a reason you can't plate serial dilutions of your culture, and then create a CFU/mL vs. OD curve for your given growth conditions?

@acvill CFU/mL is very noisy, per citation #2

From a technical perspective, for the FlopR package you mention (and I believe also for Jacob Beal's protocol) you can simply change the bead size and perform a new calibration. The only information that you need is the bead count at each point of your calibrant curve.
However, using calibration beads of a given size assumes that your cells will remain approximately the size of the beads for the duration of your experiment. We know that cell size and shape vary based on growth phase, antibiotics etc. [1] This can cause substantial misreporting of cell counts but is currently brushed under the carpet. I would suggest that if your Pichia change significantly in size or shape during their growth, it would be worth using a range of beads sizes and attempting to calibrate different growth phases of your samples against beads that correspond to cell size at during that phase.
[1] K. Stevenson, A. F. McVey, I. B. N. Clark, P. S. Swain, and T. Pilizota, “General calibration of microbial growth in microplate readers,” Sci. Rep., vol. 6, no. 1, p. 38828, Dec. 2016.

It's worth noting that the calibration of different bead sizes hasn't been formally verified yet, but there's not reason to expect it won't scale.


text:
What tests are available to detect C. Botulinum/Botulinum Toxin in fermented meats?
I am interested in the preparation of fermented dry-sausages (salamis, summer-sausages, etc) where due to the humid anaerobic atmosphere inside the salami. It is a good environment for C.Botulinum spores to grow.
The mainstream food safety practice is to use Sodium/Potassium Nitrate or Nitrate + Nitrite, which are suspected carcinogens.
I would like to prepare fermented dry-sausages without Nitrate usage and test the finished product after for the presence of C.Botulinum?
Salamis are usually 10-50cm, and if I were to consume or sell them it is viable for me to test each salami. Thus, I am also interested in the viability of taking a few cuts out of a salami to test for C.Botulinum presence, would a small cut on the sausage be sufficient, or it might be that there is a growth of C.Botulinum in a small part of the sausage not throughout the whole sausage making testing a sample not likely to reveal whether or not other parts of the sausage have C.Botulinum.
Also concerned about whether lack of C.Botulinum is sufficient proof that the salami is clear, or whether the bacteria might have died off after producing the toxin. The same concern about whether if there is toxin production should it be throughout the salami or in isolated pockets making testing a small sample from a large salami not viable.
Based on this situation what would be a test that is will allow me to detect whether a salami is likely to cause botulism?
Is it as simple as some gram stain and an optical microscope?

Isolated pockets is quite likely - so testing would be ineffective.


text:
ATP Synthase EC number
Why ATP synthase EC number is 7(Translocase)?
My textbook says it's a hydrolase but when i checked its EC number it was changed to 7.
And also ATPase(adensointriphosphatase) page that was previously classified as hydrolase is deleted in qmul.ac.uk

What textbook is this? An ATP synthase is not a hydrolase, since it does not use water to break a chemical bond (at least not in its primary function). However, it couples proton translocation with ATP synthesis. Therefore it is a translocase.
See the following passage from qmul.ac.uk what category 7 is and why e.g. ATPase is classified as translocase rather than as hydrolase. (It is a hydrolase as well, but it's not its primary function.)

Translocases (EC 7): A new EC Class
Six enzyme classes have been recognized since the first Enzyme
classification and nomenclature list was first approved by the
International Union of Biochemistry in 1961. These were based on the
type of reaction catalysed: Oxidoreductases (EC 1), Transferases (EC
2), Hydrolases (EC 3), Lyases (EC 4), Isomerases (EC 5) and Ligases
(EC 6). However, it has become apparent that none of these could
describe the important group of enzymes that catalyse the movement of
ions or molecules across membranes or their separation within
membranes. Several of these involve the hydrolysis of ATP and had been
previously classified as ATPases (EC 3.6.3.-), although the hydrolytic
reaction is not their primary function.

Thanks for your answer.I don't think it's a surprise that some "old" sources use this classification because as mentioned in qmul,ATP synthase was classified as 3.6.3 from 2000-2018.And if you don't mind i have another question:Do all enzymes that seem to belong in two classes regrading the direction that we are looking at,are classified regarding the "primary direction" that enzyme usually catalyzes?

Unfortunately, it's not that easy. There is a whole catalogue of rules for the naming and classifications of enzymes: https://www.qmul.ac.uk/sbcs/iubmb/enzyme/rules.html.


text:
DNA complementarity against reverse complementarity
I am sorry to bother with this question (i study genetics for about few hours, because I need to understand my data) and I am really confused about these two terms, because I dont know if the books uses word "reverse" interchengeably or what is going on here.
The main problem I am totally confused about are words reverse complementarity and complementarity of two DNA strands. Since I know that strands runs in antiparallel directions and two DNA strands are complementary to each other:
i.e. sequence ACTCTG is complementary to TGAGAC and vice versa.
But my question is: are two DNA strands reverse complementary to each other? Because at least in two books I have read that two strands of DNA are. But if I would apply it to my previous example and use reversion, then no way I can get to my other strand. Is the word reverse in this context use as antiparalel (5' - 3' and 3' - 5') or am I missing something and it's true that if I take the whole DNA strand, I can use complement and reverse it and it will be my other strand?
Again I am sorry to ask this really basic question, but I am so confused about this terms.
Nice day to all and thanks if someone will have time to answer my question.

There is already a comprehensive answer to a previous question of which this is essentially a duplicate.

I agree that the definitions can be somewhat confusing when first encountered.
Each nucleotide has a complement A-T, C-G. But the DNA strand are reverse-complementary because when aligned from 5'->3' they are not (necessarily) complementary.
For example:
DNA strand 5'-ATCCGG-3'
complement 3'-TAGGCC-5'
reverse    5'-CCGGAT-3'
Since we want to write all sequences in the same direction we must call the strand reverse-complementary

OK, thank you a lot. I did not realise that there is a convention to align two strands of DNA in the same direction and then speaking of reverse complementarity. But it seems like a pretty natural way for comparing. Thank a lot again, and have a nice day!


text:
Identify bush with white puff and 3 petal flowers, is it poisonous?
Is this one of the poisonous bushes I should worry about? Tiny white puff of flowers along with 3 petal flower.   What is it?

You haven't told us where you live, but it must be somewhere in the Eastern USA, as your plant appears to be the native Hydrangea arborescens.  More info available here and many other places on the internet.

That’s absolutely it!


text:
Is this flying bug swarm termites? Found in New York city, approx 7mm
Just saw a giant swarm of these fly out in between pavers and a door sill from a small crack in the ground here in New York City.  There seemed to be hundreds of them swarming out very quickly.  They are about ant-sized.  Are they termites?
Here are photos:

Yes it looks to be Reticulitermes flavipes aka the Eastern Subterranean Termite.

Welcome to Biology.SE! You are more likely to get a positive response to your answer if you explain why your identification is correct. Specifically, please [edit] your answer to add discussion of key features that led you to this conclusion and supporting references or at least validated images. Without this your answer is indistinguishable from opinion and thus not appropriate for this site. ——— You may also wish to take the [tour] and then consult the [help] pages for additional advice on [Answer] effectively on this site. Thank you!


text:
How is the surface tension of water related to transpiration?
My textbook mentions the following :
• Surface Tension – water molecules are attracted to each other in
the liquid phase more than water in the gas phase.
I can't understand how that would help in transpiration.

I think the question needs more clarity: the quote given does not fully define surface tension - it is merely a correct statement about difference between a liquid and a gas. It is also not clear why the author thinks that it plays a role in transpiration.

My textbook actually mentions three things. Cohesion, adhesion and surface tension. The first two, I can understand is to maintain the continuous stream of water but I can't understand the role of surface tension.

surface tension plays a role in liquid moving up the pores, and also in gorming droplets on the skin, but it is hard say more without knowing the context.

Is the textbook abour plants? Surface tension is inmortant in plant laefs.

Complete reference:

The transpiration driven ascent of xylem sap depends mainly on the
following physical properties of water:

• Cohesion – mutual attraction between water molecules.

• Adhesion – attraction of water molecules to polar surfaces (such
as the surface of tracheary elements).

• Surface Tension – water molecules are attracted to each other in
the liquid phase more than to water in the gas phase.

The ascent of xylem sap is aided by surface tension since it allows the water to hold together and create a continuous column of water. Otherwise, the capillarity would be affected (the water column could 'collapse' under it's own weight, for example) and plants would be unable to conduct water via the xylem. Surface tension, cohesion and adhesion are common to capillarity occurring outside of living systems as well.
Recommended read:  https://www.usgs.gov/special-topic/water-science-school/science/capillary-action-and-water?qt-science_center_objects=0#qt-science_center_objects


text:
Does the two-state gene expression model apply to constitutive genes?
Wikipedia defines constitutive genes as

a gene that is transcribed continually as opposed to a facultative gene, which is only transcribed when needed.

I don't have a strong theoretical background biology/molecular biology, and I am currently studying gene expression analytically, specifically the two-state model of gene expression, where a gene can be on the 'ON' or 'OFF' state.
From this definition I get the understanding that constitutive genes are always in the 'ON' state, although I am not sure if that is the case since other sources describe constitutive genes as genes that are expressed at a relatively constant level, which could still be a gene fluctuating between 'ON/OFF' states.

This question cannot be answered as it solely depends on the definition of OFF and ON states. I.e. is it defined by a certain protein-assembly that would attract the RNA-pol? Or does it mean that the genes is currently being transcribed? Does it depend on the DNA-accessibility? I guess your model is NOT biologically defined and instead just wants to fit some data. In this case, this question makes no sense. It's just a model and does not have to be compared to any biological 'reality'. If it fits the data, it's good!

@KaPy3141I think it very much can be answered, and your comment is the beginning of such an answer.


text:
Has anyone tried site-directed mutagenesis aimed at deleting >10 amino acids of a protein using overlapping primers?
I am trying to use primer pairs with overlapping and non-overlapping regions to create deletions within a protein sequence (https://bmcbiotechnol.biomedcentral.com/articles/10.1186/1472-6750-8-91).
I aim to delete at least 10 amino acids within a region of choice. I did PCR using the primer pairs I designed and out of 4 transformants I screened, I got one deletion I was aiming for.
I then did the same for deleting another region and I didn't get any required deletions. Vector size is ~5kb and the gene is 1.6 kb. I do Dpn1 digestion for 2 hours using 1ul of enzyume. My questions are these:
a)Is improper Dpn1 treatment the reason for failing to get enough transformants?
b) Since I am doing full plasmid amplification here, are there any things I should keep in mind while I perform the reactions?
I am terribly sorry I am not being specific anywhere. I am quite new to everything described here and has only started to get a hang of things. Any help would be greatly appreciated. Thank you!

Malka, you are on the right track with Site-Directed mutagenesis! I agree with previous answer about NEB method and their kit.   If you can, please-please, get yourself their product here.
https://www.neb.com/en-us/products/e0554-q5-site-directed-mutagenesis-kit
I have spent a lot of time with kits from different vendors, and, in parallel, attempted to combine enzymes and substitute reagents in troubleshooting steps.  All my efforts ended up as half-measures and a ultimately a waste of time.  In theory, yes, you can make it all work with off the shelf reagents, but (unless you get lucky) your substantial effort and investment of precious resources (i.e. competent cells, and time) may cost a lot more in the end.

Go for the Q5 Neb kit (all included)
Design primers using their software tool (i prefer the older version)
https://nebasechangerv1.neb.com
Use exactly as directed. And drink coffee, read papers being confident that in two weeks you will get exactly what you need.

Feel free to ask any more questions about this.
To answer specific questions you ask above:
(a) No, extended DpnI treatement should not reduce the number of transformants.
(b) Yes!  Make sure that your extension step is long enough to amplify the entire plasmid accounting for its size.  You can run a quick gel to check the size and amount of your PCR product before proceeding to the cloning step.

10 aa or 30 bases is about the limit of traditional quick-change style systems where you have overlapping or completely complementary primers.  Kunkel's 1985 paper1 used complementary primers, but variants have been developed since. The limit is partly because generating primers that don't incorporate large chunks of the target tend to form weird structures in the PCR and not anneal well; it also costs more to have primers that are about 50 or more bases long for such a PCR, compared to conventional 18-25 bases.
There is a variant of quick-change where you basically generate primers that are "back-to-back" so that you amplify around the plasmid, which doesn't limit your size of deletion. In this you use the primers to flank the region to be deleted so that you generate a linear transcript that incorporates the plasmid. New England Biolabs (NEB) has a good description of the various types here.
For these types of protocols you do need to phosphorylate the product (using a kinase such as T4 polynucleotide kinase), ligate the product using a DNA ligase and then DpnI treat the product before transformation. These can be bought as a kit or as individual components, you may even have some of these in your lab right now if it is a lab that has done cloning before.
Ref:

Kunkel TA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488-92. doi: 10.1073/pnas.82.2.488. PMID: 3881765; PMCID: PMC397064.


text:
Scientific word for organisms that can regrow themselves
Is there any term for living organisms or more specifically animals that can regrow a whole from small components (assuming sufficient quantity of cells or specific organs are present)?  For example - as I understand - worms can regrow from a relatively small part of a worm - fragmentation?  Some creatures can regrow limbs.  Many plants can grown from small cuttings or root segments (fragmentation) as well as by budding.  I wonder if any term exists to describe all of these processes, either for the respective kingdoms or more generally.  In other words is there a class of life that is definitely not like mammals, which cannot reproduce limbs let alone survive the loss of any major organ function?

Yes, there is a term: This process is called regeneration.

…and you might describe the organisms as self-regenerating.

OK regeneration is clearly a related contept, but reading up on the term it's about biological processes rather than classification of organisms. Plus humans have some regeneration abilities. Perhaps my question is asking for a false dichotomy.


text:
What are allogametes? please explain
Allo means different or unrelated I guess.Please clear me the meaning with some examples.

Where did you read the term allogamete? I dont think I've ever encountered this term. I would guess it applies to species that have a double nucleus such as many fungi species.

Here are a few notes on how to ask a question: 1) When asking for a definition, please provide the context in which you found the word to be defined 2) writing please explain in the title is useless 3) Add a space after the . at the end of a sentence.

I vote to close as unclear until your provided where you encountered this term.

@Remi.b I think you are being unnecessarily harsh. This is an example of a poorly asked question that leads to an interesting and informative answer.

@AMR I think it is still unclear whether the OP was meaning allogamy or allogamete. Simple edits would much improve the clarity of the question. Good answer though (+1)

I vote to close as it shows absolutely no research effort.

@rg255 Have you actually tried to research "allogametes?" Google scholar has 2 hits, one being a Science article from 1911. Google comes back with related hits, but none that specifically mention "allogametes." The OPs profile says that they are from India. If they are not well versed in science writing in English, then it may have been difficult to evaluate what they were seeing when not finding an exact hit. 1911 science writing would be difficult for most American science students not familiar with reading texts from the turn of the last century.

It's a poorly formulated question that would benefit greatly if the user made greater research effort, in my opinion

And....the OP should have said something by now. We've been discussing the meaning of its question for 2 days.

You may be getting confused with the term Allogamy.
Humans most higher animals are allogamous, which means that the ovum from one organism is fertilized by spermatozoa of a different organisms.
If you read the term allogametes somewhere, then the reference likely means spermatozoa from one organism of a species and ova from another organism of the same species. There were two references on Google Scholar to allogametes. This reference is in Science from 1911, no that is not a typo.
Dimorphism of the Gametes of Œnothera by Walter T. Swingle.
Species that have hermaphrodites, such as nematodes and other types of worms can be autogamous, meaning that the organism produces both the ova and spermatozoa and self-fertilize. In this case the offspring of this organism are clones of the hermaphrodite, which can come in handy in genetics experiments. Basically you are doing a self-cross.
Many of these species can also be allogamous. In the case of the nematode you usually have the Hermaphrodite, which is XX and the male which is XO. The male only produces spermatozoa and when mating with the hermaphrodite, their sperm takes precedence over that of the hermaphrodite. This is likely due to a last in first out situation in the spermatheca of the hermaphrodite. Sperm develops in the hermaphrodite first and are deposited in the spermatheca, then the ova form. If a male mates with the hermaphrodite, 'his' sperm is deposited in the spermatheca of the hermaphrodite and then as the ova pass the spermatheca on their way to the vulva they are fertilized by the sperm.
Edit
The Urologic and Cutaneous Review: Technical supplement, Volume 2 from 1914 on a section on the origin of sexual reproduction (The Origin of Amphigony), also makes mention of the term "allogametes" in reference to protist cells which are able to differentiate and fuse and then divide asexually after the fusion even. The key in this text is made from differentiated cells, allogametes, or similar cells, isogametes, that are capable of fusing and producing a new cell capable of cell division.

It is also possible that the OP is referring to allopolyploids, in which case the gametes come from two different species with different genomes that produce an offspring that can lead to a new species. This is seen in plant breeding. If OP responds with clarification I will attempt to expand on that.

license: Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/3.0/


text:
What structures does our brain use for a quick indexing of all the data we store in our heads?
What structures does our brain use for a quick indexing of all the data we store in our heads?
From how my brains work (i.e. that I can very quickly recollect something from the past) I would say that my brain does not do a linear scan of all the memories, otherwise it would take a lot of time to recollect something. So, we for sure have indexing structures in our head.
From the time complexity point of view I would say it should be at least logarithmic or even faster. Do we maybe have B-trees or LSM-trees implemented in our brains?
I am curious to learn more about the topic, but so far I was not able to find a good explanation about it.
I am playing around with creating my own database and would like to try to simulate the workings of my brain to get something similar. Hence, the curiosity in the question.

B-trees!  No computer programs apply.  Our brains are essentially entirely parallel and asynchronous, unlike any nontrivial machine man has ever built.

We don't know..

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

@Community, please, edit your comment to limit it to a specific problem with enough detail to identify an adequate course of actions for updating my question. Thanks.

Brains are not computers, and do not process information like computers.
The trees from CS you are talking about are ways to run linear information stores through a central processing unit. Even with machines that can sort of do these operations in parallel, they are still very much series computations.
Brains, on the other hand, are massively parallel, and information is encoded in a very high dimensional space, not linearly. There are no "bits" in a sequence to sort through. Information does not just move from "storage" to some "processing" part of the brain, it is constantly being processed at all stages in all places. When a memory is active, that information is linked to all the other related information physically because neurons are connected to each other. That is, when you think of a "cat", you might retrieve a memory of a specific cat you saw in a specific place because there is a physical connection between some of the same neurons that are active when you think of "cat" and the neurons encoding that engram of the specific cat memory. No "search" is necessary; every extremely high-dimensional brain state is followed by another extremely high-dimensional brain state. When you "search" your memory for cats, you are just biasing these future states to be those which involve neurons that were involved in the past during experiences of cats.
I think the most attractive models for understanding how brains index information are attractor networks. There are in silico models and implementations of these sorts of networks, but without the parallel hardware of a biological brain they aren't particularly efficient.


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Why do chromosome pairs have different shapes and sizes?
When I look at microscopic images of human chromosome pairs I see that they have different shapes and sizes.
Is there a deeper biological reason for that? Is there some evolutionary pressure for them to be this way?

Please clarify your specific problem or provide additional details to highlight exactly what you need. As it's currently written, it's hard to tell exactly what you're asking.

Chromosomes are different sizes - they have different numbers of base pairs in them. That's why they look different...

@MattDMo They are curved at different angles. Why are the angles different? Why are they not all straight lines?

Because the nucleus is crowded, and there isn't enough room for all of them - especially the longer ones - to be stretched out straight. If you haven't already, please read the Wikipedia article on karyotypes.

Think of the chromosomes as a bunch of ropes of different lengths (i.e. size in base pairs). if you quickly try to sort out a bunch of ropes on the ground with a long pole from a platform 50 feet up (basically how karyotype images are created), do you expect the ropes to be straight, or for some of them to have some wiggle?


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Why are small land vertebrates less common than small invertebrates?
There are some very small vertebrate species. The dwarf chameleon is a very cute example. But why are vertebrates on that scale so uncommon compared to invertebrates such as arthropods? Warm blooded creatures have issues with their metabolism as they get tiny, but even cold blooded vertebrates seem to be lacking at this scale.
At first I assumed that maybe it had something to do with the amount of time that they have had to evolve. Land vertebrates have been around for a bit less time than land arthropods such as insects, so maybe insects just has more time to take advantage of the niches that come with being that tiny. Looking in the ocean, we can find more small species of fish, and some large invertebrates such as crabs and their relatives, large clams, etc which might support that idea.
I have no idea if this is true though. Are there any biological reasons for why tiny vertebrates, on the scale of a few cm in size, are so uncommon?

I can think of a couple of answers.

Vertebrates are generally soft since they possess an inner skeleton and animal cells do not have cell walls. In order to survive falls from a larger height or other mechanical stress, a hard carapace would help tremendously, arthropods already do it and fill the niche by having an external skeleton of chitin (lancelets can exist since water offers little mechanical hazards when compared to the land environment). Hard reptile skin is a contender, however, it may not be sufficient.
Animals, such as chameleons which you've described, are staggeringly different when it comes to their innards as compared to arthropods. The differences lie in the respiratory system, circulatory system, the way they excrete, reproduce and so on. As an example, an arthropod cannot have lungs, instead it uses a network of canals in it's body in order to exchange air. Reptiles have lungs, which is why their size is limited as the respiratory system cannot be downsized forever, and it's deevolution can be considered impossible.
Smaller vertebrates cannot exist since arthopods and other animals prove to be superior in speed and resilience in their size niche, eliminating any small vertebrates which downsize.


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What kind of insect eggs are these?
I was looking at one of my forgotten old cotton T shirt inside my wardrobe and I saw these spiky things protruding coming out on it's collars and sleeve ends. I tried to pry some off them out but they're really stuck, like it's glued on. I tried to break on of them and the piece slightly had a bloody color inside. I think they are insects eggs, maybe I'm wrong? If they are, should I be worry about these? Like probably infestation problem? Are they still in incubation or are they just empty leftover shell eggs? They're size is like 5mm or so.

Curious indeed. Are you certain that it's not an artifact of manufacture, a weighting added to the hem like is done on curtains with lead-shot? Reason I ask, they look like seeds to me - but maybe someone knows better. They do seem awfully odd, even spacing, all in the hem like that. Was there any evidence of similar structures elsewhere in the wardrobe or on that shirt?

Well, I'm sure they're not seeds or that someone put it inside the collar. Seems to me that whatever it is might came out though the fabric. It's just a simple cheap cotton T shirt and inside the collar they have just an elastic.

Well, I'm stumped. They don't resemble the eggs of the regular clothing moth, or any eggs that I've come across - to me they honestly look like seeds of some sort. I hope that you get an answer to this strange and intriguing puzzle. If you get it off-site then please pop-in and update the post with an answer, this'll stick with me until I know what it is.

hmm..doesn't have to clothing moths while inside the wardrobe. I dry them on outside hanged on clothes line to sun dry. Maybe something came and put them and lay their eggs while they were drying. I'm still sure they ain't no seeds. I'll find and try other sites and if I did, I'll sure to update you on. I hope they don't hatch and become infestation of not.

At least if they hatch they'll be easier to identify! (A few wash-cycles should probably have rendered them inert I imagine).


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Neutralization Assay: Comparision of IC50 values between different viral loads
I've been performing Neutralization assays for HIV pseudovirus against a dilution series of different antibodies, and was wondering whether the viral load influences the measured IC50.
My specific reasoning: I apparently understand the concept of normalization (0-100% neutralization). But if you calculate the virus titer beforehand (e.g. 1000 RLU/uL), and add exactly 10'000 RLU viral load (10uL virus) to every well, wouldn't you get a different IC50 value for the same virus with the same antibody when you use e.g. 20'000 RLU (20uL virus), because in this scenario you would need more antibody (thus higher IC50) to achieve a 50% neutralization for the 20'000 RLU wells compared to the 10'000 RLU wells.
What I don't understand: Apparently the difference of viral loads can be resolved by normalization, but this I don't understand. Shouldn't you "always" get a lower IC50 value for the same virus with the same antibody dilution series when you add less virus (smaller viral load). Doesn't this mean that you can't compare different IC50 values between different assays and viruses if you don't know the original viral load? (e.g. smaller IC50 values could be explained by smaller viral load instead of higher affinity of the antibody to the virus)
Thank you for clarifying.

Yes. This is one of the basic concepts in virology, and presumably in other branches of biology too. It doesn't just apply to antibodies; it also applies to chemical (drug) treatments. You need to apply a standardized concentration of the virus(es) across your treatments or you can't compare them.
A good example of this is the classic influenza assay HAI (HemAgglutination Inhibition), which is a test of sera for the presence of antibodies inhibiting hemagglutination and therefore response to influenza infection, requires you to standardize the HA (Hemagglutinin) content of your viruses before performing the assay via the HA assay.
For other viruses you might use a different standardization process, perhaps something like a TCID50 (Tissue Culture Infectious Dose 50% endpoint) or a plaque assay used then to work out that X infectious units (in TCID50/ml or pfu/ml respectively) is a good point to work at for your antibody assay.

Thanks for the answer!


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Does the rate of change of blood pressure depend on cardiovascular fitness?
For example, going from sitting to standing can raise blood pressure. Does the rate of that change depend on cardiovascular fitness? Like, does a more fit person reach their standing blood pressure faster?

From personal experience, sadly now long ago, my then 30 bpm heart rate generally resulted in a slow change in blood pressure as monitored by slight dizziness on standing rapidly.

@JonCuster Orthostatic hypotension AKA postural hypotension is the term for the delay in blood pressure change with standing up. Incredibly common in the general population; about 25% people have it once over 60, though less than 5% under 45. I've been in a similar situation when younger and more athletic than I am now.

@bob1 - correct, the point I was trying to make is that a slow heart rate leads to a long time for feedback. The time it takes me to stand up is less than 2 seconds, and if the heart contracts every 2 seconds it can be a long time, subjectively, to stand there feeling woozy.


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optimum amount of pH buffer for an experiment
If trying to do an experiment involving the effect of pH on enzyme activity, is there any sort of guide as to how much pH buffer to add?

There's no way to answer this - it entirely depends on your assay and how you want to go about it. Further details needed.

@user338907 Can you please make your comments into a full answer? Thanks.


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Circular definition of female and ovum?
The Britannica defines the egg (ovum) as "the female sex cell, or gamete." It then defines "female" as "If only eggs are produced, the reproductive gland is an ovary, and the primary sex is female". The two definitions are circular.
If a new species which can produce two kinds of sex cells is found, what is the exact rule to determine which is an ovum or sperm? By size (the bigger is the ovum), by shape (the round one is the ovum), by movement (which travels in the medium is the sperm, but what if both can travel), or else?
Species have various ways of reproduction. For example, both sexes of marine mussels release sex cells into sea to fertilize, so the conventional intuition of sex (who bears the offspring is female) is not enough.

Just found it's already answered here.

In that case you should delete the question or request closure.


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How do animals obtain energy by consuming other organisms?
This is a question I never really thought about much, but now that I have, I realized it wasn't explained in detail before. Maybe it is irrelevant, but how do animals get energy by consuming each other? Specifically,there is a certain text I am wondering about: "The hare uses a significant fraction of the assimilated energy just being a hare -- maintaining a high, constant body temperature, synthesizing proteins, and hopping about. This energy used (lost) is attributed to cellular respiration.The remainder goes into making more hare biomass by growth and reproduction."
This confuses me in several ways.

What differentiates "just being a hare" and the functions associated with that from growth and reproduction? Don't all of those processes include cellular respiration?
Later in the article, it states that the conversion of the assimilated energy into new tissues is what provides food for foxes. How does this happen? Is energy stored after conversion?
Basically, I don't exactly know how to word my question, but hopefully this provides enough information. If not, please let me know. Thanks!
Edit: Also, please keep in mind that I just started high school and don't have a good grasp on this concept yet, so it is likely I won't understand some responses.

"Energy" in foods come from macronutrients: fats, carbohydrates, and proteins. These are the primary physical components making up the mass of an organism (the "hare biomass by growth"). Making these molecules requires a lot of energy; some of that energy becomes stored in chemical bonds. It's possible to retrieve some of that energy by breaking them down into their component parts, all the way down to carbon dioxide, water, and nitrogenous wastes.
If you eat a hare, you digest the proteins, carbohydrates, and fats that make up the structure and energy storage of the hare.

How does cell division and growth fit into that, if at all?

@user386598 Well, an organism of growing and dividing cells is going to be made up of more and more of the organic molecules of life.

This makes sense, thank you.


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What exactly causes the measurable voltage shown in an ECG/EKG?
I am familiar with how an ECG works (measuring current to determine voltage), with heart physiology, and also the mechanism of muscle action potentials. A lot of sources describe the action potential as perpendicular transmembrane flow of ions in and out of the cell. However, what confuses me is that an ECG is able to record the "flow" of the action potential across the heart muscle. Wouldn't this mean that there is some ion flow in the direction of the AP? And not just perpendicular ion flow in and out of the cells during de/repolarisation?
What I assume happens is that what is actually being measured is the small flow of ions leaking across the gap junctions between myocardial cells.
Can someone clarify how ECGs work and what it is actually measuring? Much thanks.

Took electrocardiography 30 years ago,  As I recall, the value on an EKG at any time and across any lead is the voltage difference between the two points (different leads).  There is no general movement of ions across the heart from one lead to another.  The The contraction of muscle cells near one lead do contribute to depolarizing adjacent cells though.  It does raise a couple physics question though.  I'll review and see if I can formulate an answer.

What you're referring to here is recognized by your ECG as mean operating values a commonly accepted as a one constant of the Conduction Systems that exist in the heart and brain. Through the sodium ion channels of the tissues of the involved organ. Operating correctly ekg,ecg values are tuned for the recording and reception of that minuscule amount of electrical current flow. More or less is recognized as a functioning deficit in your patient. Electrolytic deficit categorized needing an influx of Electrolytes depending on patient injuries and imposing symptoms determines level of infusion.
ie; synaptic tissues,over axons to dendritic tissues. I'm going to query it,through a friend a Cardiopulmonologyst. Get back afterwards.

This is completely incomprehensible. I do not think you understand the question asked.

Actual people don't speak or write like this; even folks with flight of ideas make more sense than this does. Hopefully it's an AI generated answer. A bad AI generated answer to boot. (Use a different site.) -1. (Although reading it made me chuckle, so there's that.) ["Electrolytic deficit categorized needing an influx of Electrolytes depending on patient injuries and imposing symptoms determines level of infusion. ie; synaptic tissues,over axons to dendritic tissues." lol!]

I'll give this the benefit of the doubt and say that it's an absolutely  awful translation of a potentially good basis for an answer. Please [edit] to prove me correct, hopefully adding supporting references as you go.


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Can consuming durian together with alcohol harm or even kill you?
In Singapore and Malaysia (and possibly other countries), it is a well-known "fact" that you shouldn't consume durian together with alcohol because it can kill you.
This is sometimes dismissed as a myth or urban legend (e.g. 1, 2).

Singh (1941) reported

mysterious deaths occurring sometimes following the consumption of durians and alcohol.

Maninang, Lizada, and Gemmaa (2009) suggest a possible mechanism:

The durian–alcohol anecdote has been sheltered with over 250 years of belief. Few attempts to validate the interaction have vaguely disproved the alleged antagonism between durian and ethanol (Nilveses & Seangsirinavin, 1986; Ogle & Teh, 1969, 1971; Techapaitoon & Sim, 1973). None of these in vivo studies, however, considered the possible interference of some components in durian on the enzymatic system for detoxifying alcohol. To our knowledge, this is the first attempt to explore the molecular basis of the durian–alcohol anecdote and no other literature has yet linked a fruit to adverse reactions that may arise upon interaction with alcohol. ...
yALDH significantly lost (P < 0.05) enzymatic activity in the presence of durian fruit extract at all concentrations used

Ho and Bhat (2015):

the sulphur content of durian is reported to have inhibitory characteristic on ALDH activity and can be mortal in person who consume durian and drink alcohol simultaneously

So, can consuming durian together with alcohol harm or even kill you?

There's a related post on our [medicalsciences.se] site, Should one avoid consuming durians when drinking alcohol? which pretty much answers this one.


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What makes the measles vaccine so effective compared to other vaccines?
The measles vaccine (administered alone or as part of a combination vaccine like MMR) seems to be incredibly effective on a range of criteria, namely:

Frequency: 1-2 doses required during lifetime.
Age: Safe to give to young children (e.g. the NHS gives the first dose at 12 months), so there's a limited window for infection before vaccination - and particularly before children start to mix at school.
Effect: Reduces the likelihood of infection to close to immunity.
Duration: Effects appear to be very long-lasting, possibly
life-long.

Many other vaccines seem to perform worse on at least one of the above, e.g. they require annual doses, reduce the severity of symptoms rather than the likelihood of infection, or can't be given to very young children.
What is it about this particular vaccine (and the virus it targets) that makes it so effective, even though it was developed before some breakthrough technologies such as mRNA?

First, it's not just the vaccine: immune responses just to measles itself are themselves strong and long-lasting, and reinfection is rare (see https://en.wikipedia.org/wiki/Measles). My understanding is that it is not entirely known why exposure to some pathogens results in longer lasting immunity than others, but there are some known contributing factors.
One factor is that from the perspective of the immune system, some pathogens are not really one pathogen but a group of them that all look different to the immune system because they have distinct surfaces; we call these major groups serotypes. For a virus like influenza, there are many serotypes. For measles virus, there is just one: if your immune system is able to recognize one measles virus it can recognize them all. Even though there is genetic variation in measles, the surface parts recognized by the immune system are quite constant.
Further, antibodies recognize several distinct sites on the measles surface glycoproteins. Changes in just one of these sites don't really help the virus escape immune detection, so there isn't much natural selection for those mutations.
Another, related, factor is the host. Humans are the only host for measles virus. Relatively widespread vaccination of humans means that there is a limited population for the virus to reproduce and create new variations in. Other viruses that affect humans can spread and mutate in other animal hosts and then spread back to humans. That can be a mechanism to eventually accumulate mutations that make the virus's surface very different from the original strain and therefore difficult for the immune system to detect.
In summary, the things that make the measles vaccine effective are really just traits of the measles virus. We struggle to make effective vaccines against viruses that change a lot, that don't themselves evoke long-lasting immunity, or that use specific mechanisms to disable or evade the immune system.

Thanks, that's really useful. One minor follow-up: to what extent are these offset by measles being a very contagious / infectious virus (e.g. it seems to have the highest R0 of most common viruses, as well as requiring a high vaccination threshold to eliminate)?

@pwaring You could maybe ask that as a separate question but I think it's too complicated to expect a simple relationship either direction. For example, the extremely high transmissibility of this measles virus could be one reason it's relatively stable: any changes that reduce transmissibility are going to have difficulty competing even if they come with other immune-evading benefits.

Live attenuated vs inactivated or "subunit" vaccines
There is a key difference between measles and influenza vaccines. Measles is a live attenuated vaccine (it's a live virus), just like yellow fever virus and these vaccines provide the very best protection, which is widely considered to be life-long. This has been challenged with yellow fever virus, but it is nevertheless highly efficacious
Influenza vaccine is an inactivated vaccine so the presentation to the immune system is impaired.
Most vaccines are not attenuated because it's risky, i.e. if they revert they'll cause an active infection and thereon can be transmitted. Polio is an exception, which is attenuated, and offers superb immunity.
Moreover, when a virus and attenuated vaccine virus enters a cell it undergoes a conformational change when its infecting the host cell (all viruses do this) and antibodies that bind to the virus at this stage of the life cycle will point blank block infection, i.e. they stop the virus entering the cell. If a virus can't enter the cell - it's dead, the technical word for this is "neutralised". An antibody just binding to the virus doesn't necessarily stop the virus infecting the cell and in limited cases it can enhance its ability to infect (antibody dependent enhancement). An inactivated vaccine has a lot of difficulty correctly undergoing a conformational change to "lock onto" the host cell - it's already dead prior entering the body.
A live vaccine generates a massively better immunity, exposing more of the proteins used in its life-cycle to antibodies and a lot more neutralising antibodies. Antibody memory can be life-long.
I'm not mentioning cellular immunity because that has a shelf-life (arguably six months, probably longer).

Genetic diversity
The issues about low genetic diversity of measles are also valid. Measles is a bit like holding the same two cards in a poker hand - every hand, sooner or later it can't win because everyone knows what the cards are.
Influenza's genetic diversity is really large, it's a bit like choosing from two suites of cards in a poker hand and the solution can be any combination. Thats hard to memorise (for the immune system). Specifically, influenza in particular flips/changes is antigenic coat making it difficult for antibody memory between infections to be effective. Influenza's potent trick of 'flipping', measles can not do. Influenza has a segmented genome and can switch its protein coat by "mixing" its two different key surface proteins involved in the infection life-cycle ('mixing theory').  Its technically called "antigenic shift" and measles can't do it because it has a single stranded RNA genome.

See the bibliography for further details especially Oxford's "Human Virology".

Bibliography
"Human Virology" by John Oxford, Kellam and Collier by Oxford Press Fifth Edition 2016
See chapters on Polio, Measles and Influenza
This is text is the most comprehensive virology text at graduate level virology and covers all the material described above. The only downside is there is no sixth edition. Yellow fever virus is poorly covered here and the vaccine is omitted. Yellow fever virus is in the chapter Flavivirues.
Fields Virology: Emerging Viruses 7th Edition 2021
by Peter M. Howley MD, David M. Knipe PhD, Sean Whelan
This is more specialist, but covers SARS-CoV-2 and yellow fever virus, which are not really covered in the "Human Virology" by John Oxford.
For in depth genomics: Principles of Molecular Virology by Alan Cann  is a standard text, but "Human Virology" is sufficient ESPECIALLY for influenza. John Oxford was an influenza investigator

Comment polio vaccine
@JenserCube raised the issue of Polio live attenuated vaccine. This is really a separate question but what they are requesting, I'd prefer to avoid talking about because criticism of the polio vaccine got mixed up with conspiracy theory and of course the current health czar for the USA is a vaccine skeptic.
There is a recent trend toward replacing the polio vaccine with a "subunit" vaccine (the old term), basically a reverse genetics based vaccine. A number of publications are proposing this. I'm personally not aware of a current phase 2 trial (the minimum requirement to say there's a series alternative). Thus I would simply say the polio vaccine has been incredibly successful and arguably came close to global eradication (in which case we'd no longer need a vaccine). So my personal view, whilst aware of the counterpoints, is stay the course, hold our nerve and get shut of this infectious disease once and for all.
I am well aware that this is an active research base towards an alternative vaccine, but my personal view is hold the course of global eradication, because once thats done - polio virus is gone the same way smallpox was erradicated.
I honestly can't compromise a long-term, heartfelt viewpoint for a few upvotes or down-votes particularly moving into an era that could turn decisively 'anti-vaccine'. One current vaccine at phase 3 I helped build the molecular foundations of it, so I've a track-record in reverse genetic vaccine design.

It would be helpful to include reliable sources supporting your answer, as there are some mistakes here. This is why answers without sources are considered poor answers.

Polio has some of the issues you list precisely because of this. As @anongoodnurse says it would be nice with references for each claim.

Addressed above

Fair point about conspiracy theory. That was definitely not the road I wanted to go down and I am by no means trying to argue against the effectiveness of the polio vaccine.

I'm a bit confused by 'Most vaccines are not attenuated because it's risky' - given that measles can cause severe complications (including disability and death), why would we give an attenuated vaccine for measles and not other less severe viruses? Or is it the case that measles is so dangerous to unvaccinated that the trade-off is worthwhile?

Hi @pwaring its a good question, but a separate question outside the scope of this answer. Yellow fever BTW has an 80% mortality rate for non-Africans and that is a really good vaccine as well.