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What are the characteristics of a promising drug?
The drug development industry is a multi-billion dollar economy powered by a multitude of research bodies from industry and academia. A big risk is that drug development costs are in the tens of millions of dollars when looking at the most successful drugs
Generally speaking, what makes a good drug for research for those researchers working on these projects? Why are some potential drug pursued and others not? What traits, according to the experts, justifies such a vast investment?

Just a note, not all drugs are proteins.  Some are biologicals, others are small molecules, some are even mixtures of compounds.  This can make a difference, as I've heard it said that biologicals are sometimes expected to work better but are more likely to be toxic or have off-target effects.

Sadly no.  That advice was mostly word-of-mouth and from my notes.  Here's a link that somewhat implies support for that theory but it's not specific: http://www.bio.org/articles/how-do-drugs-and-biologics-differ

The specific characteristics sought after will vary widely, but that being said, there are a few key features many groups look for.  I've talked to and heard a number of individuals at a number of different companies, and there are a lot of different techniques and pathways looked for.  In general, however, the single, most important factor is value.  If the drug won't make money, it's almost never considered "promising."  Maybe that's a sad fact of the way the world works, but it's a fact.  The average drug costs around $1 billion (USD) to bring to market so the successful ones damn well better recoup that (Orphan drugs and companies actively pursuing them often have an easier time of this, as the FDA is understandably less restrictive and fast-tracks their progress).
That's not actually so simple.  It's a large, crowded market out there, and there is a lot of intellectual property floating around.  A wise businesswoman told me that her company focuses on the middle-ground.  A target that is too promising is probably already owned by Genentech, so they aim for the ones that are below the attention of the giants but still quite profitable.
After that, it's mainly what you might expect.  High avidity, long half-life coupled with low toxicity (more on this later), small size (if possible), and a realistic time frame.  A month can mean millions of dollars, so working for a decade is not necessarily a possibility.  Often, companies will use high-throughput screens of thousands, even millions of small molecules to look for an effect.  The screening process is usually extremely rigorous, and many potential candidates are eliminated as not good enough.  Sometimes they do some fancy stuff such as 3D modeling but it is definitely a field where brute force can still be successful.
Often those screens are used against primary cells to test for toxicity.  In fact, throughout the entire process, toxicity is a huge factor.  A full 20%-40% of drug attrition is due to safety and liability issues.  It's a constant balance: nothing can risk the health of healthy, control volunteers, but sometimes more risk is acceptable.  The first HIV/AIDS drugs were incredibly toxic, but that was okay because it was still better than the alternative.  These days, the toxicity requirements for HIV/AIDS treatments such as HAART are stricter.  Short-term antibiotics are allowed more risk since you only take them for five/seven days, whereas long-term medication for chronic illnesses are held to much higher standards.
Basically, loss of eyesight might be preferable to death, but it's unacceptable if treating the flu.  The mantra I've been told is three things:

Don’t treat one thing but cause another
Don't cause cancer
Don’t cause birth defects or alter the ability to have children

Toxicologists are conservative, willing to throw the baby out with the bathwater.  In short, Fail Early.


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How to download different kinds of data from NCBI eutils?
I have been researching NCBI eutils and wish to get some 'big data' from it. I know that I can construct queries to query one of (I think) 8 databases, like this:
https://eutils.ncbi.nlm.nih.gov/entrez/eutils/einfo.fcgi?db=nucleotide&id=2765657
which queries the nucleotide database for an id 2765657.
I am looking for a query that will return:

genetics sequences.
functional annotations of the sequences (e.g. exons, introns)
metadata (e.g. chromosome)

Would anyone be able to show an example of how to search the eutils to get samples of records as described above?
I would need to know:

Which database to search?
how to list available records?
how to query for a sample of the records?

Any help would be greatly appreciated.
Many thanks.

The Entrez module in Biopython is able to do all the task written here.

thanks Shred, I am aware of this module - but have not tried it yet. Would it be able to help me in determining which database would be best and which filters would be useful? Many thanks

I am looking for complete protein coding gene sequences, from one organism, preferably expressed in close temporal and spatial proximity, that have have quality annotations (e.g. exons, introns, codons). I have no particular preference on the model organism. Just looking for something to exercise LSTMs on.

ok I got a gff3 file from GENCODE - this looks good

Downloading all this using an E-utils search would be cumbersome as you would have to search against multiple databases. For your specific needs, a GTF/GFF3 file would be good. Basically, a GTF/GFF file has genome annotations (positions of features) and includes features like exons, CDS and in some cases even special codons (such as that for selenocysteine). This file would not have the sequences but you can retrieve the sequences from the corresponding genome sequence fasta file using the co-ordinates described in the GTF file. There are several software tools that can do this; for example gffread and bedtools-getfasta (you can even write your own script which would not be very difficult).
You can download the GTF and the corresponding genome fasta files from these sources:

ENSEMBL (many organisms)
NCBI-RefSeq (many organisms)
UCSC genome browser (many organisms)
GENCODE (for human and mouse) and modENCODE (for Caenorhabditis worms and Drosophila)

You should look at the details of the genome assembly. Different releases/sources may have different extents of curation.

source: Stack Exchange

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Chromosome picture, how to interpret?
In this guide i was reading for annotations, there is a diagram of chromosomes of D.mel, I am confused as to why the chromosomes are all attached to one another, is this picture taken in a certain stage of mitosis in which they are all condensed?
Original source
 (page 2).

You need to look up polytene (giant) chromosomes.

Polytene chromosomes are over-sized chromosomes which have developed from standard chromosomes and are commonly found in the salivary glands of Drosophila melanogaster. Specialized cells undergo repeated rounds of DNA replication without cell division (endomitosis), to increase cell volume, forming a giant polytene chromosome. Polytene chromosomes form when multiple rounds of replication produce many sister chromatids that remain synapsed together.

You can read more about it at wikipedia (http://en.wikipedia.org/wiki/Polytene_chromosome)

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Trouble understanding overlapping genes.
I read the wikipedia passage on overlapping genes.
https://en.wikipedia.org/wiki/Overlapping_gene
The Tandem overlaps they speak of make sense to me, as I understand that their are different reading frames(3 in total if I understand correctly) for genes to be read in the 3' to 5' direction and that this causes an out of phase overlap.
This out of phase tandem overlap for genes was shown by the picture below:

I am having trouble understanding the other scenarios spoken about in the passage, the in-phase tandem overlap and the Anti-parallel strand overlaps.
Maybe I just lack a good enough background in genetics.
I do not understand how you can have an in-phase tandem overlap. If the genes were in phase, how could they ever overlap and be different genes? Wouldn't they both respectively have a stop codon, and then one stop codon would "stop" the translation?
I picture it like this: pardon the crude drawing

The anti-parallel strand overlaps also confuse me as well, for I do not understand how genes can overlap when on opposite strands. If the genes are on opposite strands wont that mean they wont have any overlap in the first place? Am I missing something basic? Thanks.

A gene has got nothing to do with a stop codon. As you might know there are non-coding RNAs as well and the genomic regions responsible for transcription of these RNAs are also called genes.
Genes that have an overlap in their genomic locations (without considering the strand orientation) are called overlapping genes.
In case of same strand overlaps, the genes should have a different transcription start/stop site. Alternatively spliced RNAs are not considered overlapping transcripts. In certain cases, transcripts with an alternative transcription start/stop site are not considered separate genes but rather, isoforms, especially if the gene produces an mRNA and the alternate transcription start/stop sites just affect the untranslated regions (UTRs) and not the coding region (have a look at this article).
Your confusion is quite valid and gene nomenclatures need some redefinition/clarification.

Regarding the in-frame overlap, I think of the genomic region like this:

SA--SB--AA--BB-AAA-AA--CC-C--A---AS---BS

Where

SA = Start gene A.
SB = Start gene B.
- = Non-coding or non-transcribed part of DNA.
A = transcript region for gene A.
B = transcript region for gene B.
C = transcript region used by A and B.

So in that example the primary transcript of gene A will consist of 13 regions. Gene B has 7 regions. They overlap in-frame in 3 regions (C).
Another more simple example could be where gene A is a subset of gene B:

SB,B,B,C,C,C,C,C,B,B,BS

Where the first and last C regions are the start and end regions of gene A.

Just for completeness sake could you post an example of an anti parallel strand overlap? I think I have an idea of what one would look like in my mind but I am not too sure.

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Drosophila biarmipes gene annotation, transposable element?
When doing an annotation of a contig31 of Drosophila biarmipes genome, the gene predictions(in orange, red, green, tan, blue, brown, and gray) picked up numerous possible genes in the area circled in blue below:

So according to the GEP(genomic education partnership) guide I was reading, I was to then BLASTP the amino acid sequence from these predicted genes to a non-redundant protein database. Using NCBI BLAST I did just that, and observed alot of results of significance (E value < 1 X 10^-5).
Many of the BLASTP results were somthing like:

Reading up on gag proteins I found they were related to transposable element activity, specifically reverse transcriptase activity. So then should I conclude that these regoins are showing a gene because they are a Transposable element, or the remenats of one?
I also found:

And reading on Wolbachia I found that there is Horizontal gene transfer with its genome and Drosophila in general, according to wikipedia:
http://en.wikipedia.org/wiki/Wolbachia#Horizontal_gene_transfer_and_genomics
So then, can I conclude the area circled in blue is a result of Transposable element, and not an actual gene belonging to D. biarmipes?

Do a nucleotide BLAST first. This seems to be just a partial match. You cannot conclude that this is a transposable element. Also see if RNAseq data is available.

@WYSIWYG        THe RNA seq data is in the first picture, under the blue circle, there are black peaks that correlate with RNA seq data, the first is labeled "RNA-Seq alignment summary for mixed embryos". As for the nucleotide BLAST, i did a tBLASTn, since I only had an amino acid file to work with. The results were still a same link to the pol gag protein. The title of the highest significance match was: "Drosophila ananassae retrotransposon putative gag-pol protein gene" .


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Can a gene be split between different genomic locations several kb appart?
Can you have exons and introns of the same gene separated by hundred of kb in a genome? If so, how is the full mRNA assembled in such distance?
I'm working on plant mitochondria and I've seen an annoted gene that is cut between very distinct part of the genome.

Is the sequence of this plant mRNA in the public domain or one you have determined yourself. If it is in the public domain you should give a citation.

The isolated exon that led me to this question is nad5 in Arabidopsis thaliania mitochondrion genome (GenBank accesion NC_037304.1). I came accross this related question link and the word "trans-splicing" led me to a paper from 1991 reporting its discovery PMC453077. I still can't understand how these pieces can be put back together amongst all the other pieces available.

Well, yes, the GenBank data shows the nad5 gene to have three exons, quite a distance apart. Plant mitochondria are rather specialized, so it sounds as if you will need to read some specific reviews on introns and splicing in that system, assuming that you have read up generally on RNA splicing first.

Are you asking if transcripts with introns above length 100 KB exist? Or are you asking if a gene can be separated by a DNA-stretch that is not transcribed?

Yes you can have exons and introns of the same gene separated by hundred (even thousands!) of kilobases.
Here is an example for the human genome:
"On average, there are 8.8 exons and 7.8 introns per gene. About 80% of the exons on each chromosome are < 200 bp in length. < 0.01% of the introns are < 20 bp in length and < 10% of introns are more than 11,000 bp in length."
Source: https://www.ncbi.nlm.nih.gov/pubmed/15217358
For the splicing process, it is not really a problem for the distance. Let me reframe the questions by challenging the assumption you are making: long distances in a sequence correspond to long distance in space.
Actually, DNA/RNA molecules are can have a lot of tertiary structure for example, G quartets for DNA, hairpin structure for RNA.

Source: https://upload.wikimedia.org/wikipedia/commons/5/5d/ATPC_secondary_structure.jpg
Long sequence distances are a poor indication of the real distance in space. Thus although from an engineering point of view it is incredible what molecular splicing can achieve mechanistically speaking. As far as I remember, the length for the intron/exon is important for the efficiency/timing of the splicing process (I might be wrong here on the specifics).

The averages you quote are for human genomic DNA. The question is about plant mitochondria.

Indeed there are for human genomic DNA. I would suggest that you re-read the question: the person work with plant mitochondria, but the question is not about plant mitochondria. I interpreted it as a more general question to which I gave a general answer to help the person identify the knowledge gap. I have edited the answer to include this precision thanks for the suggestion.

Thanks for your help, it's a good reminder to keep in mind the tertiary structure. I now realise my title was unclear. My interogation wasn't about the total lenght of a gene but more about having only an exon of gene really far from the other exon (without having an intron in the middle but non-coding DNA or other genes). I came across the idea of trans-splicing, I think it's what it is? Then, how does the spliceosome knows which pieces come together in a single gene?

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Proteoglycans vs Glycoproteins

On the left, is my histology book, on the right there's qoura (the internet).
My histology textbook says that "unlike glycoproteins, proteoglycans have attached GAGs which often comprise a greater mass than the polypeptide core"
What I found on qoura is "The carbohydrate content of proteoglycans (10-15%) is far less than glycoproteins is (50-60%)"
I tried googling the question but couldn't find the answer so which of these is correct ?

Campbell Biology (11th edition) states that proteoglycans are up to 95% carbohydrate.

More context would be helpful, but it's possible the data from the Quora post was incorrectly transcribed from a table on this biotech site, which states that the carbohydrate content of proteoglycans is 50-60%, compared to 10-15% for glycoproteins.

However, the first comment on that page suggests that the table was initially incorrect:

Hi Anne,
This is a great post, but the numbers for the carbohydrate content are reversed. It’s 50-60% for the proteoglycans and 10-15% for most common glycoproteins (and things like uromodulin and most mucins would be 30+% sugars).
Best,
Jared Gerlach

So that may be the source of confusion, here.  It seems the commenter is an expert in the field of glycan research, so the table is likely correct.

Was the table edited afterwards ? Because the table shows 50-60% carbs in proteoglycans and 10-15% in glycoproteins which the writer and the commenter agree on

Yes, it seems that is the case.  I've edited my answer to reflect that, thanks.


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How can an animal live without a head / brain?
The human body is unable to do anything if it loses its head - as the brain is separated from the body, the body immediately dies.
But why does the same not happen to other species or animals? (As they are somehow similar organisms/living things, like humans). However, when the head is cut off from a chicken, snake ( also insects, but it's different system, answered here... I mainly interested in bigger animals) ... the body continues to move. Why and how does this occur? Who manages the functionality of body, if there is no manager (brain)?

There is nothing different between animals and humans. Have you tried to google Why do headless chickens run??

@Remi.b - Have you googled "Why do headless humans run?" Of course there's a difference.

@anongoodnurse My comment was misleading. Sorry about that. I reacted to the use of the term "live" as if in animals some fake essence of life of the organism was not dependent on the presence of the head while it would be in humans.

related: https://biology.stackexchange.com/questions/79167/can-turkeys-run-around-when-their-head-is-cut-off-like-chickens-do

In general, and this is somehow speculative, the animals that you mention have more autonomy in their extremities and less control from a head, muscles have evolved autonomous responses perhaps as an evolutionary advantage, ( i.e. having a brain be in charge of reactions can add precious milliseconds ), there also seems to be a relationship in between brain size ( specific motor control areas) and this autonomy, which introduces a metabolic cost (i.e. our big heads need a lot of energy)  Further, there are other biological considerations for providing extremities with their own resources and thus to a degree be more independent, perhaps another evolutionary strategy. At the other end of this control-autonomy relationship, the head takes control of the extremities most of the time and their interdependance is greater, so if you cut it no movement will happen, although we still possess a few hard coded reflexes that work for a while provided they have enough energy left or a blood source.
To give you an example: a cat ( somewhere in the middle ) can still walk when you cut the connections from the brain /head  to the legs due to reflex actions ( the cat has to be on a treadmill though).

Some limb movements in vertebrates can occur at the level of the spinal cord (e.g. swimming behaviours in fish; coordination of alternate movement of legs in walking animals). This might support some uncoordinated movement for brief periods following complete severing of the head.
However, most (all?) vertebrates require nuclei in the brainstem to control breathing. Without an intact brainstem the body will not survive.
Decerebrate cat walking on a treadmill (but with an intact brainstem): https://www.youtube.com/watch?v=wPiLLplofYw
Chicken survives without a head (but with a brainstem): http://modernfarmer.com/2014/08/heres-chicken-can-live-without-head/

Brain is needed for lung breathing motion (at least for mammals, maybe for all vertebrates), which limit the life span without brain.
Attention: In the case of the chicken, the low stages of the brain are at the level of the "neck", so sometime cutting the head is not fully cutting the brain and some base functioning can keep working.


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Are children's ears more vulnerable to loud noise than adults?
I am interested , what are child's ear development stages?
Do newborns' have already developed (defensed) ear like adults have and there is no much difference with regard being vulnerable toward sudden burst noise, which we (adults) bear easier (i.e. popper burst within 1 meter range)?

I would suggest that this type of question, not relating to specific biological processes, might be more appropriate on SE Medical Sciences.

@David sorry, but there is no chance such question to be accepted on medicals.se. I have experience there. my question is formed to be non-medical and it is about ear-development stages.


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Does anyone know of a study supporting predictions #2 or #9 of the Extended Evolutionary Synthesis?
Regarding the 12 predictions made by the Extended Evolutionary Synthesis (http://extendedevolutionarysynthesis.com/about-ees/), does anyone know of a study that supports #2 or #9?
It seems like #1, #3, #5, #8, #10, #11, and #12 do not violate the modern synthesis.  #2 and #9 would though.  Agreed?  But I know of no study that supports them.  Not sure about #4, #6, or #7.

Note that the title ask for a study that support two given predictions while the content of the post (and your comment below my answer) ask whether the two given predictions given predictions from another set of knowledge. You might want to clarify that.

I'm essentially asking two questions in the post.  A primary question:  whether there's a study supporting #2 and #9.  A secondary question:  whether it violates the modern synthesis.  I think I overlooked the distinction the EES authors are making between genotypic variance and phenotypic variance and that #2 and #9 are probably exemplified by facilitated variation.

There is no debate
The so-called Extended Evolutionary Synthesis (EES) is just a new term for a whole lot of knowledge that roughly speaking everyone acknowledge to be true. EES is really just an extension but does not say anything that conflict with previous theory. There is no conflict and no contradiction.
The only debate stand upon whether or not our current understanding of evolutionary processes deserve a new name or shall we just stick with what we call the modern synthesis.
Shall we use the term ESS?
I talked about this renaming with about 20 evolutionary biologists and all of them consider such renaming useless. However, by the end of the day, they all mainly did not care much.
My personal opinion about the renaming
IMO, one reason such naming is silly is that there has not been a major work that have completely and suddenly changed our understanding. We just keep discovering more and more as we always did. All the new things we discover we just used to integrate it what we already called modern synthesis. Those advocating for a renaming define the modern synthesis not as most people do (that is everything we know about evolution so far) but as the set of knowledge that existed in 1950.
It is funny that even the discovery of DNA did not bring about a renaming of our theory of evolution as we only considered that it added up to our current understanding. I doubt that the emergence of new fields (such as niche construction and its heritability or developmental noise) would be in any way sufficiently revolutionary to cause a renaming. And I say so even though I also work in the field of mutational and developmental noise.
Unclarity
The points listed in the link are rather unclear and it is therefore impossible to comment on them. Take the #2 you are interested in

heritable variation will be unbiased

The term variation has no strict definition. The term variance is clearly defined. However, a variance in itself cannot be biased, only the distribution it comes from can. So, this simple sentence mainly makes no sense (to me at least). If it does make sense to those who wrote this list, then, OP, you will need to provide explanations from other source or ask for its meaning before asking for its validity.
Further reading
This paper will offer you opinions from both sides. As implicit from the above, one side (namely, the "counterpoint" side as referred to in the paper) seems to me to represent the vast majority of evolutionary biologists.
Addressing your comments

Thank you for the clarification. I think by "heritable variation," the authors mean mutation. You feel it's still true that they are unbiased?

"mutation" is not a variable, it does not follow a distribution. It cannot be bias or unbias.
Mutational effect on fitness is a variable and follows some distribution which is most definitely biased (deleterious mutations are much more common than beneficial mutations; usually modelled with a double gamma distribution; see here).
Mutational effect on the phenotype is a variable and follows some distribution. It may well be biased indeed. The distribution of mutational effects on the phenotype were mainly unconsidered by evolutionary biologists who contributed to place the first bricks in the modern synthesis (Fisher, Wright, Haldane, Muller, etc...).
These "early" evolutionary biologist never made predictions about the distribution of mutational effects on the phenotype that would contradict today's predictions. They just had little understanding and did not talk about it much.
Again, in your question you are opposing EES with Modern Synthesis as if they were to be opposed. But by doing so, you need to clearly define Modern Synthesis. By default, one just consider the Modern Synthesis to include all the knowledge we have in evolutionary biology and therefore by definition, elements like niche construction, biased mutational effects of phenotype and other field of knowledge that one might want to specifically associate to EES only.

Do you still feel it's true that natural selection is the only source of the evolution of adaptation?

This question depends only on the definition of adaptation. If, for example, you consider a lineage being poorly adaptive becoming adaptive as the environment changes to match their phenotype (and not the other way around) to be an adaptation, then typically nobody would have never thought that only natural selection would cause adaptation. I would personally be happy to call that an adaptation.
The question is not a question of evolutionary process but only a question of the definition of adaptation.
Note, as an extra source of confusion, when talking about adaptation one may refer to the changes yielding to higher fitness (whether the change is genetic or not is up to you) and/or to the resulting state of having a high fitness.

For example, if there is an epigenetic modification that is transgenerationally stable and adaptive, is it an adaptation?

Again, it depends upon what you call adaptation. I would be happy to call that an adaptation.

the discovery of DNA would not cause a reconceptualization of the modern synthesis because it was consistent with it.

Everything from the EES is consistent with the modern synthesis. Most of it are just add-up subjects that brought little consideration before. Some of it might break some simplified generalization that may have been done before though. Many does affect our view of the grandeur and complexity of evolutionary process but none is contradicting what we previously thought.

what you mean by "becoming more adaptive as the environment changes." Would you give an example?

Imagine a population is perfectly adapted to live at an environment where the temperature is 18˚C. The current temperature is 15˚C though. Let's imagine a case where the population does not change much through time (eventually due to a lack of genetic variance to select from, eventually due to a low mutational variance or a recent bottleneck) but the environment is getting warmer and after some time the environment is at 18˚C. The population is now perfectly adapted.

I appreciate it and I've already read the paper.  Irrespective of renaming, the question is whether those predictions violate the modern synthesis.

The answer is "No, because nothing in the EES contradicts the modern synthesis. EES is just an extension (but most people don't really like the idea of renaming our theory based on this extension)" I edited my answer. Hopefully that will clarify my point.

Thank you for the clarification.  I think by "heritable variation," the authors mean mutation.  You feel it's still true that they are unbiased?

Also, do you still feel it's true that natural selection is the only source of the evolution of adaptation?

For example, if there is an epigenetic modification that is transgenerationally stable and adaptive, is it an adaptation?

Also, the discovery of DNA would not cause a reconceptualization of the modern synthesis because it was consistent with it.  If anything, it confirmed the principle of the modern synthesis that inheritance occurs through genes.

@sterid I addressed your comments in an edit in my answer.

I appreciate your view a great deal.  Thank you.

"This question depends only on the definition of adaptation. If, for example, you consider a lineage being poorly adaptive becoming adaptive as the environment changes to match their phenotype (and not the other way around) to be an adaptation, then typically nobody would have never thought that only natural selection would cause adaptation."  I do not think it's purely a semantic issue.  It is also the conceptual issue of which definition for adaptation making sense.   I'm also not clear about what you mean by "becoming more adaptive as the environment changes."  Would you give an example?

Re "becoming more adaptive as the environment changes" : Is this what you feel occurs during niche inheritance?

@sterid See edit

I see.  That part is clear now.  Thank you very much.


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Can the value of heritability be greater than 1?
Heritability defined as genetic variance divided by total variance seems to be bounded between 0 and 1.  However, I see a way of calculating heritability on this page (http://www.radford.edu/~rsheehy/Gen_flash/Tutorials/Linear_Regression/reg-tut.htm) with:  2*Covxy / Varx.  However, a result calculated in this way could be greater than 1.  What am I missing?

I am missing where on that site it is stated the formula you are giving is calculating heritability.

Where it says "Calculating the slope," but I multiply by 2 for a sexual population, according to the last paragraph on the page.

@skymningen It's at the bottom: "Regression of offspring on only one parent underestimates narrow-sense heritability by about 50%. Therefore, Christine's analysis suggested a narrow-sense heritability of 2 x 0.414 = 0.828." The problem is that if you had both parents, the slope would be closer to 0.828, and it wouldn't be doubled.


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What's the difference between shotgun sequencing and clone based sequencing?
In a lecture during  my undergraduate degree we were introduced to the race to complete the human genome. Celera were competing with Sanger and collaborators to sequence the human genome. Celera wanted to patent some sequences, whilst Sanger et al. wanted to keep their findings in the public domain. Celera knew they couldn't compete with Sanger et al. with conventional cloning techniques which required a lot of man-power, "so instead opted for shotgun sequencing".
To me it seems like both clone based sequencing and shotgun sequencing could have been used together, however the lecturers semantics were a bit vague (...okay, I wasn't paying close enough attention!) and I didn't ask for clarification at the time.
Whilst I appreciate this is a large topic, what are the main differences between Celera's shotgun sequencing and Sanger's clone based sequencing during the late 1990s? Are "shotgun cloning" and "clone based sequencing" actually mutually exclusive terms?

Umm, you realize that an answer to this will be quite a few pages long right? Why don't you look up some of the common sequencing technologies on wikipedia and post a more specific question?

What a coincidence, I happen to have had the exact same question, also from a lecture during my undergraduate degree, within the same context of the human genome project. I suppose the lecture notes haven't changed that much...

The v short answer is that in shotgun sequencing, the sequencer is fed a set of random sequences from the target.  This can be done for instance by mechanically shearing DNA and then building a set of shotgun sequencing jobs which are then compiled back together by a genome assembler (i.e. in whole genome sequencing projects) into a full sequence.
In clone based sequencing, the DNA to be sequenced is specifically sectioned off into sequence copied into of plasmids (BACs - bacterial artificial chromosome) to divide the sequencing job into sections- a divide and conquer strategy.  BACs can be 150 kb long just to give you an idea.
These are not mutually exclusive terms; clone-based sequencing projects usually use the large clone libraries to section the project into a set of shotgun sequencing projects.

In short: the basis of the process is the same in both, the difference relies that in the clone based sequencing you make DNA library of the pieces of DNA clones you got from the sequence you used in first place. Like that, data management and the mounting of DNA contigs will be a lot easier computationally speaking.
In shotgun sequencing the same is done but without the cloning and libraries, you directly mount contigs from sequenced sequence pieces, thus better computers are needed.
Also I think your terminology is wrong: shotgun strictly means the sequencing based on the random shearing and consecutive construction of contigs. I think you better mean whole genome shotgun sequencing with "shotgun sequencing"(as the clone based sequencing also is a shotgun sequencing method).

"To me it seems like both clone based sequencing and shotgun sequencing could have been used together,"
I believe they were.  The public project cut up the genome into BACs and shotgunned every BAC, and then put the BACs together, while Venter used some sequence position information from the public effort to help him place his shotgun pieces.
https://en.wikipedia.org/wiki/Human_Genome_Project


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Using an analytical balance (0.0000 precision) to measure µg (micrograms)?
If i needed to prepare 200µg/ml of proteinase K, and the proteinase K  was in a solid powder form, would I  have to weight out 200 µg using an analytical balance, and if so, is it possible with a balance that only goes up to four decimal places? (i.e. 0.0000) I assumed 1 microgram is 1×10-6 gram and therefore 0.000001 gram.

How is this a biology question? If you are interested in the finer points of using a balance, you might enjoy reading this: A question of balance

As others have said, that mass is too small to measure with a standard analytical scale. There are two options you could use:

Make a larger volume of your 200 ug/mL solution.
Make a concentrated stock such as 20 mg/mL. This a 100X stock; you can make 1 mL of 200 ug/mL proteinase K with 10 uL of stock and 990 uL of buffer. Small volumes are easier to measure than small masses (with a micropipette).

I guess i still am having issues with the concepts of concentrations. It is just such a hard concept to wrap my head around, i wish i could have practice with it.  For your first option, making a larger volume, what would that entail? The recipe requires 200ug/ml , so i wouldnt know how to deal with anything else. For the second option, how would i go about making the 20mg/ml stock? Would I measure out 20miligrams of the powder = 0.020grams on the scale? And then mix that with 1 ml of water? But even with that more concentrated stock, i dont understand how i go from the 20mg/ml to the 200ug/ul

@RoSiv You should practice. I imagine your teacher could help you and there are many resources on the internet.

In this case make a large quantity and store it as stock - this is a general lab practice. Preparing 10 ml of your protK solurion would you need 2000ug (=2mg) of protK powder.


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How to make µg/ml concentrations of proteinase-K?
How does one prepare concentrations in the mass/volume (weight/volume) form, for substances like nucleic acids or in this case, proteinase? A detailed example would be helpful.
I need to prepare squishing buffer for a DNA extraction /PCR exercise to help me learn the materials better for a in class "internship".
The squishing buffer recipe I found from a laboratory class manual describes it as: 10 mM of Tris-Cl, pH 8.2, 1 mM of EDTA, 25 mM of NaCl , and 200 µg/ml of proteinse K (freshly diulted).
I understand molarity concentrations well enough. One only has to determine the total molar mass of a substance, in this case NaCl, and with that molar mass (g/mol) then use the given desired molarity (in this case 25 mM of NaCl, which equals 0.025 mol /one liters) to get the number of grams of the substance needed. In this case, since we need 0.025 moles / 1 Liter, we would need to multiply that by the molar mass to get: (g/mol) X (mol/l) = g/l of NaCl.
I understand percent solutions (to a degree) as well. If I  needed 1% agrose gel, I would take 1 gram of agarose solid powder and bring that to 100 ml total to obtain a 1%, either using water or buffer?
I am having trouble understanding the concepts of concentration, in reference to mg/ml.
From what I understand, µg/ml is a weight (mass)/volume type of concentration. Therefore, should it be the case that if I wanted to prepare
200 µg/ml of proteinase (freshly diluted), how would I go about it? Would I add 5 ml of deionized water to get a working volume of 1000 µg of proteinase per 5 ml of water, which would be equivalent to 1 mg of proteinase per 5 ml of water. From that 1mg of proteinase to 5ml of water, should I then draw 0.2 µl (or 200 ml) of solution to then have the 200 µg/ml? Or am I totally wrong? I tried googling for more information, but I became more and more confused.

What is a "squishing buffer"? The composition seems to indicate some kind of protease lysis buffer, but I have never heard of anything by that name.

@MarchHo              Yes it is a buffer used to break down the flies protiens via the protienase. Since I will be doing PCR on flies.

@canadianer      So lets say I  have some of that lyophilized powder. Could you give an example of doing so? I just cant seem to picture doing the process for some reason.

Using your agar example, your agarose is 10g/L = 10mg/mL.

What is NaCl₂ ??

It depends on what form your proteinase K is in.

You may have a stock solution of some concentration, in which case you just add a specific volume according to $C_1V_1=C_2V_2$.
Example: Let's say you want to prepare 5 mL ($V_2$) of 200 µg/mL ($C_2$) proteinase K from a stock of 1000 µg/mL ($C_1$). You're looking for the volume of stock solution to add ($V_1$).
$$V_1=\frac{5\ mL\ \cdot \ 200\ \mu g/mL}{1000\ \mu g/mL}=1\ mL$$
So you take 1 mL of the stock and bring it up to 5 mL.

Or you may have a lyophilized powder. In this case, you have two options:
1) You can make a stock solution from the powder and, again, use $C_1V_1=C_2V_2$. This has the advantage of not having to weigh the powder every time you need to use it. This may not be a viable option, however, as I'm not sure of the stability of proteinase K in solution.
2) You can just weigh out the amount that will give you the desired concentration (which depends on the volume of buffer you're making).
Example: Again, you want to prepare 5 mL of 200 µg/mL proteinase K. The total mass you need is:
$$200\ \mu g / mL\ \cdot \ 5\ mL=1000\ \mu g$$
So weigh out a milligram and bring it up to 5 mL.

There's nothing special about mass concentration; you can convert it to molarity given the molecular weight of proteinase K. According to Wikipedia, proteinase K is 28.9 kDa or 28900 g/mol. If you are making this conversion, however, make sure you find the actual molecular weight of the protein you're using. 200 µg/mL is equal to:
$$\frac{0.2\ g/L}{28900\ g/mol}=6.92\ \mu M$$


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Do animals demonstrate target practice (i.e., playful accuracy behavior)?
Do animals demonstrate any sort of accuracy-motivated play? (please provide examples!)
Consider most human sports and related hobbies. Most share the common goal of hitting a target or being accurate, and in the case of organized sports, points are typically awarded for being accurate. Archery, firearm past times, football, soccer, basketball, hockey, baseball, tennis, billiards, bowling, darts, beer pong, horseshoes, and countless other examples all are based on competitive accuracy.
Humans seem to be obsessed with accuracy in our competitive past-times. Examination of more ancient sport interests demonstrate less preoccupation with accuracy (though of course there is variation between cultures -- see here or here, ), and perhaps just more with demonstrations of strength and dominance. However, competitive accuracy was alive and well in ancient times: boxing, sword fighting, lacrosse, hoop rolling, golf (and Chinese equivalent Chuiwan), and others already listed are just a few examples of accuracy games with more ancient origins. It seems, then, that the human species has  had a fascination with accuracy for a long time.
However, do animals have the same (or really any) preoccupation with accuracy? I've seen countless examples of animals 'playing' or competing (e.g., pets & captive animals playing with toys & enrichment objects,  young males of various taxa sparring or wrestling, whales playing with their food, monkeys sheep herding on dogs, etc.), but is there any evidence or example of wild animals playing 'games' based on accuracy?

No need to comment on reasons why animals play. I understand that the bulk of animal 'play' is preparing them to be successful adults & that accuracy would typically not be immediately relevant for most animals. (Though bears trying to catch salmon, chameleons trying to catch insects, & osprey diving for fish are just a few examples where accuracy is necessary). In these cases, 'practice'  would not really be competitive or playful tests of accuracy, but rather just failed attempts to gather food. I'm curious if more playful examples of tests-of-accuracy exist in the animal kingdom?

I guess it is unlikely that playful tests of accuracy really developed before the evolution of hands and feet that are more amenable to throwing objects. Still, I could imagine instances where accuracy games involving spitting, rolling, fishing, insect-catching, etc.  could exist

Nice questions, but I voted to close. The questions are each quite broad individually, and you stack three distantly related queations on top of each other.  Please ask specific questions, one at a time. In its current form it's too broad to even start a partial answer attempt. Also, consider other SE sites. E.g., the history of human play is not on topic here.

I guess so yes, just develop them in independent questions with the appropriate background info targeted to each question. Simply copy-pasting the same text twice wouldn't be too constructive.

Voted.  Good luck!

Voted to reopen - though I'd suggest the question would benefit from reducing the amount of text... right now it's fairly substantial reading, that can put people off reading it properly...

One example that comes to mind is archer fish. There is evidence that they improve with practice, but they practice on targets that are food items, so I'm not sure whether that counts as practice with respect to your question. Interestingly, there's also some evidence that they can improve their accuracy simply by watching other archer fish succeed.

Does this count?
https://www.telegraph.co.uk/news/worldnews/northamerica/usa/2508756/Dolphins-play-with-bubbles-in-Florida.html

The playful creatures create the bubbles with their blowholes, then
  chase and swim through them.


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Why do fruit flies go so close to large reservoirs of liquid when they are likely to fall in due to surface tension?
I had accidentally left out half a glass of red wine by the windowsill when I went to sleep. As anyone knows, if you do this with a window cracked you will get quite a few fruit flies near it in that time(region dependent obviously).
I noticed that many of them had simply fallen in as there was nearly nothing on the sides of the glass. What causes this behavior in a likely death scenario? Is it only exhibited during extreme hunger? Would this also not likely be an evolutionary cost or is that outweighed?

Fruit flies are attracted to the smell of alcohol (~rotten food). Large reservoirs of liquid alcohol in which they could drown seem rare in nature. There are some (selectable) reasons why flies are drawn to the smell of alcohol. Anecdotally, never seen a fruit fly drowned in liquid water suggesting that they cope fine with more frequently occurring liquids.

@tsttst Even just the time we've been industrialized would be reasonable to start to be negatively selected for in a species like this no?

Might as well ask why they like to land on solvent-borne paint... and permanently get stuck in it. I suppose they can fly off the surface of liquid... under normal circumstances.

As indicated by their name, fruit flies can be found on fruits, where they prefer to lay their eggs. Since the larvae feed not on the fruit itself but on microorganisms found on rotting material, they will especially target overripe fruits. Therefore fruit flies have evolved to identify and respond to odors emitted from ripe fruits. Red wine similarly emits odors that are attractive for fruit flies. Since fermentation is a form of controlled rotting (in both cases material is decomposed by microorganisms), the resulting compounds can be similar, which includes ethanol and 2-phenylethanol.
So, fruit flies are attracted to the smell of wine, that's why they approach the glass. Due to their small size and the surface tension, they should be able to land on water without drowning. But wine isn't water. With an ethanol concentration of 12% we see a reduction in surface tension from ~72mN/m to ~45mN/m, which is comparable to adding some detergents.

This explains, why the flies are not able to escape and drown after landing. I would nevertheless argue, that the selection pressure isn't strong enough for an evolutionary adaptation. In their natural environment, they usually would not encounter large bodies of liquid which they are attracted to and which are not able to hold their weight.
In evolution and behavior we have to consider the entire species. While a few unlucky animals will die in wine glasses, most other flies will not. For them being attracted to the smell of overripe fruit and ethanol is an advantage. It's a cost and benefit calculation. For the species, the costs of a few individuals dying is outweighed by the enormous benefit of finding a good place to raise their offspring (laying eggs into rotting fruits). Therefore, the attraction to these smells and approach are positively selected for, even though this results in death in wine for some members of the species.
Watching fruit flies at a trap, you see that mostly flies land on the container rather than on the liquid, indicating that they already avoid touching down on liquid. On the other hand, fruit flies don't seem to be very acrobatic, with a part of their landings being "crash landings". Hence, some of these flies might just accidentally end up in the liquid or by walking down to the edge of the liquid and being caught up by it when touching it. Even though I couldn't find hard data on this, "landing on liquid reservoirs" doesn't seem to be necessarily a strong behavior in fruit flies, that evolution could negatively select for.

Although this is from  a popsci article, a biologist says that fruit flies can normally land on liquids and take off from them thereafter:

However, simply leaving a shot glass full of fruit-vinegar will only serve to feed the flies. The surface tension of the liquid [Ed. note: You remember surface tension, right?] allows fruit flies to land on liquids but not drown.
To break the surface tension you’ll need a tiny bit of dish soap. Dish soaps contain surfactants, which are molecules with a head that likes water, and a tail that doesn’t. Because of this property, the surfactants tend to situate at the interface of air and water, forming molecular “cliques” that disrupt the surface tension of water.
By putting a tiny bit of dish soap on your finger, and touching it to the surface of the vinegar in the shot glass, you will add enough surfactant to break the surface tension. (Add too much and the molecules in the dish soap will alert the fruit flies to danger).
Now fruit flies will unknowingly attempt to land on the vinegar for a little sip, then, before they can react, will break through the surface tension and begin to drown.

So if this is true/confirmed, then they do have enough of an evolutionary adaptation to the [surface tension of the] usual liquids of their diet. Just not to [trace] dish-soap contaminated liquids.
And I can confirm from a peer-reviewed paper that dish soap is indeed used even in science trapping experiments:

The cup trap (figured by Beers et al. 2010) is a 946-ml clear plastic
drinking cup with a clear plastic lid (Solo, Urbana,
IL), with four 1-cm-diameter holes in the sides of the
cup near the top. A drowning solution or drowning
solution plus bait was placed within the cup. Forty
grams of boric acid and 0.5-ml liquid dishwashing
soap (Palmolive Pure and Clean Spring Fresh Dishwashing Soap; Colgate-Palmolive Company, New York, NY, USA) were added to a batch of 3.8 l of
water or aqueous bait to make a drowning solution
used in all traps. Boric acid served to inhibit microbial
activity of baits and captured insects, and soap
reduced surface tension to promote capture and submersion of trapped insects. For both types of traps
and all treatments in all experiments, each trap contained either 300 ml of drowning solution or 300 ml
of bait in drowning solution

0.5ml of soap to 3.8l of water is very little, but that's all it takes to drown the little flies. It seems entirely plausible to me to have this level of trace contamination on glasses that have been dishwashed.
And as a complementary survival mechanism, fruit flies can apparently survive 12 hours of drowning at room temperature (or more in lower temperature):

Flies submerged at 23°C did not survive after a submersion time of 12 h whereas flies submerged at 3°C survived up to 72 h of submersion.

@Frieke may be on to something, namely that ethanol lowers the surface tension of the solution (with water) by itself, significantly. At 20-25 degrees C, pure water has a tension of about 72 mN/m. Adding 10-15% ethanol lowers that to about 47-42 mN/m. Also, the trapping science paper does mention that

Very few flies were captured in traps baited with 10% ethanol in water.  Traps baited with a combination of acetic acid and ethanol in water captured more SWD flies than traps baited with acetic acid or ethanol solutions alone.  These results indicate a synergy of the two materials, and of the two chemicals, as lures for SWD.

Interestingly wine has substantially higher attractive effect than an equivalent ethanol solution though:

Wine is about half as effective a vinegar and about a quarter as effective as a combination of wine and vinegar. On the other hand, a 10% ethanol solution was 20-50 times less effective than alcohol 10% plus acetic acid 2%. So there's more to wine than ethanol that attracts the flies.
So the flies probably do avoid the higher alcohol concentrations... unless there's also vinegar.
Another paper finds that wine's surface tension is mostly determined by its alcohol content.

So it does look like:

Fruit flies are fairly strongly attracted to wine (on the same order of magnitude as to vinegar).
The surface tension of wine is substantially lower than that of water.

I'm still not sure what the surface tension of (2%) vinegar might be, in comparison. Pure acetic acid has substantially lower surface tension compared to water, about 27 mN/m at room temperature. And if Wikipedia is correct, 10% acetic acid solution has about 55mN/m around temperature. So acetic acid lower the surface tension less than ethanol, but not by much less. On the other hand, fruit flies are much more attracted to acetic acid than to ethanol (alone). So perhaps that's enough to explain why they drown easier in wine.
Actually it look like Wikipedia is wrong here. From an authoritative source, acetic acid has nearly the same effect as wine on surface tension; at 11.4% it has 43.5 mN/m; just 3.2% is enough to lower the tension to 55.6mN/m; at 1.6% it's 61.7mN/m.

So probably just the fact that acetic acid is much more attractive to flies than ethanol is the only factor (other surfactants excluded). Perhaps they "confuse" a higher concentration of ethanol to a lower concentration of acetic acid, which they would be able to handle as far as surface tension goes.
Yet another source (actually quoted in the previous one, on the next page) gives even lower values for ethanol surface tension.

Apparently determining surface tension is a hard problem, both experimentally and theoretically. So I'm not sure we can properly compare figures reported in different sources. Alas no single experimenter has done both ethanol and acetic acid in the same paper/setup. But what was observed about their relative attractiveness to flies is probably sufficient...
And for ballpark comparisons, a similarly or lower surface tension as that of 10% ethanol (or acetic acid) i.e around 30-40mN/m can be obtained by a adding a few ppm (parts per million) of a surfactant, such as LAS or AE.

And as related fun fact, it's sex-starved males who prefer alcohol, in fruit flies too! https://www.nature.com/news/rejected-flies-turn-to-booze-1.10227

I appreciate this answer but it doesn't really address why something like this hasn't been and isn't being negatively selected for. For example, one could easily imagine adaptations by which melanogaster evolve aversion to certain liquid smells of those in which the suface tension cannot support. This could even occur via simple reinforcement. Wrt the work on psuedo suspended animation in fruit flies this makes sense as a general survival mechanism but I wonder why a more nuanced model has yet to be selected for.

@hisairnessag3: not enough evolutionary pressure for that until now, apparently, at least at the trap concentration. See https://biology.stackexchange.com/questions/35532/why-do-some-bad-traits-evolve-and-good-ones-dont The first quote does mention that they can detect the detergent at higher concentrations. Perhaps some lab experiment will [try to] evolve a line of flies in that direction.

0.5ml in 3.8l is a 0.013% solution of soap. In half a glass of wine of, let's say, 1/8l=125ml, that would account for about 16microliters of soap. I have not tried this, but I would guess you would taste that if there is that much soap left on your glasses after washing. Therefore it does not seem plausible to me that soap contamination levels would be high enough for drowning the flies. Do you have any idea about surface tension of alcoholic solutions compared to water?

@Frieke: apparently alcohol has lower surface tension than water: https://en.wikipedia.org/wiki/Tears_of_wine That might also explain it, since the science paper also mentions that high alcohol concentrations don't attract flies much. Small amounts of alcohol mixed with vinegar have a better attraction effect. And since alcohol also seems to affect the nervous system of the flies (see 1st comment), it's possible they also get "drunk" which might contribute to them getting drowned easier in the presence of alcohol.

@Frieke: However, the wiki link also says that in an alcohol solution the alcohol evaporates faster at the surface, so the surface tension of the solution is probably close to that of the solvent (water).

Thanks for the edit. I still think you can delete or shorten the soap part, since this is in my opinion not relevant for the issue. you have covered the attraction to wine (and fair enough, some of it might have turned into vinegar over night) and the lower surface tension that would contribute to the flies drowning, but you haven't touched on the evolution part of the question.


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Can environmental pressures affect genes in one generation?
Environmental pressures are the catalyst of evolution. Pushing a species to adapt to changes therein. My question is can these mechanisms cause significant adaptation over one generation(parent-> child). My thinking is the pressure of the equivalent of human trauma in a species(depending on neurological and biochemical makeup of said species) would be enough to induce genetic influence.
If not, what is the minimum theorized timeframe for environmental pressures to act?
Edit: This is not a duplicate, the previous question is about one animal's lifetime

I've tried to answer your question, though I am a little confused about the sentence re: "the equivalent of human trauma".

@DeNovo to clarify further, an event that would cause some sort of psychological ailment as a result(i.e PTSD,GAD) in humans. From what I understand, things like anxiety from social or non-threatening situations are neutered versions of the physiological reactions we had pre-society.

@hisairnessag3  so your point is that if parent in childhood experience some trauma ( for example sexual abuse) if that change genetic information hand over to children?  Am i correct ?

I voted to close as "unclear what you're asking", because the question, though unclear, is certainly not asking if an individual animal can evolve after birth. It is asking evolution can occur in one generation. Indeed it can, and only ever does.

Don’t think that this is a duplicate as the main question (ignore speculation on trauma)  can be answered with a definitive YES. This was demonstrated recently for first time: Rowan D. H. Barrett et al., “Linking a mutation to survival in wild mice.” Science 363, no. 6426. (February 1, 2019). doi: 10.1126/science.aav3824

@tsttst I believe the first time it was demonstrated was 160 years ago...

The formal link between going from environmental pressure to change in frequency of a naturally occurring variant of a gene within one generation for non-laboratory condition is new. Given everything else known about evolution and molecular mechanisms of inheritance this is expected. The above shows it explicitly within one generation. 160 years ago they did not study genes.

Can environmental pressures affect genes in one generation?

Yes. Environmental pressures that cause a change in allele frequency will always produce changes in a single generation. If the environmental pressure is present for more than one generation, it will affect changes in allele frequency over a longer time span.
I'd encourage you to look at this set of case studies from UC Berkeley's Understanding Evolution
To clarify what "pressure" means in the context of evolution. Selective pressure, or evolutionary pressure, as wikipedia describes it, is anything that changes the reproductive success of an organism. It is, by definition, something that has an effect in one generation. Selective pressure causes organisms with certain heritable characteristics (and certain alleles) to reproduce at a different rate from organisms with other heritable characteristics (other alleles). The pressure selects for certain alleles.
Considering the comments, it sounds like you're describing a disorder (psychological ailment, e.g., PTSD, GAD), which may have a heritable diathesis, and an environment (trauma) that causes the expression of that disorder. To consider that environment to be selective pressure, it would need to be present at a population level and cause individuals with that heritable diathesis to reproduce at a different rate than individuals without it. If it did, you would see changes in allele frequency over one generation. Indeed there is no way for selective pressure to cause a change in allele frequency without it occurring in a single generation.  I'm not aware of any study that has demonstrated this to be the case for any anxiety disorder.


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What was the biological mechanism that allowed Tarrare to eat so much?
Are there any conjectured mechanisms that cause Tarrare's extremely oversized stomach and abdominal cavity? Along with his superhuman appetite of course.  Whether from a medical perspective or a genetic one (I.e a potential diagnosis that is perhaps quite similar or the genes that would be at play).
Even correlated genes and their subsequent mechanisms that are being affected could be an interesting addendum.
*I would like to make sure that any answer is also trying to explain the enlarged esophagus and abdominal cavity(seen during autopsy) along with the appetite, sweating, and other observed manifestations of an underlying condition(see Death section of Wiki for info and associated source material in book "Two-headed Boy and other Medical Marvels").

Typically questions about symptoms and medicine are closed. Since this is a famous case study, I think it should stay open. It is, however, a historical case study meaning that there will be speculation about a definite cause. Personally, I think there is enough evidence to discuss biological concepts of disease in relation to Tarrare, but I understand why there are votes to close the question.

@James yes, this is why I also tried to focus on the genetic aspects involved. If you'll look I updated my post to also try and get some more info on the deformities seen at autopsy. If you want to expound upon those I will go ahead and select your answer.

It's a good question, and I like the idea of considering this as a genetic disease case study. I've found what I can from the source material on the autopsy.

Recently there was Mr Mangetout also... he ate a plane. https://en.wikipedia.org/wiki/Michel_Lotito#Death

@LifeInTheTrees I don't think Mr Mangetout is a useful comparison, he was purely a snowman, if perhaps one with a psychological eating disorder, and didn't consume excessive quantities of digestible material as Tarrare did or share any of Tarrare's other symptoms, Tarrare's daily consumption of vast quantities of ordinary digestible food coupled with a lack of any obesity and the reported high body heat suggests some form of metabolic condition,, was

Tarrare had these symptoms:

Grossly increased appetite, eating raw meat and other inedible things
Excessive sweating with bad body odor
Chronic diarrhea, which was "fetid beyond all conception"
Wide jaw, abnormally large esophagus and stomach
Loose skin around his cheeks and stomach
Heavily stained teeth
Unusually soft fair hair
Apathetic temperament

He didn't vomit excessively and he was not overweight.
Description of his skin and internal organs:

When he had not eaten, his skin would hang so loosely that he could
  wrap the fold of skin from his abdomen around his waist. When full,
  his abdomen would distend "like a huge balloon". The skin of his
  cheeks was wrinkled and hung loosely, and when stretched out, he could
  hold twelve eggs or apples in his mouth.
At the autopsy, Tarrare's gullet was found to be abnormally wide and
  when his jaws were opened, surgeons could see down a broad canal into
  the stomach. His body was found to be filled with pus, his liver and
  gallbladder were abnormally large, and his stomach was enormous,
  covered in ulcers and filling most of his abdominal cavity. (Wikipedia)

Possible causes:

Constant hunger and excessive eating was obviously due to chronic diarrhea that resulted in loss of most nutrients he consumed, which also explains why he was keeping relatively low body weight.
Saggy skin, large esophagus and stomach can be explained by a combination of eating large amounts of food at once and lax connective tissue - the later could be genetically predisposed. It is known that eating huge amount of food at once can result in acute gastric dilatation.
Enlarged liver (hepatomegaly) and gallbladder were likely due to abscesses as part of purulent infection in his abdomen discovered at autopsy.
Chronic diarrhea could be caused by a gastrointestinal infection or intestinal parasites, which may also explain his other symptoms: eating inedible things ("pica"), excessive sweating, bad smell of the skin and stools, apathy (due to anemia) and ulcers and pus found in the gastrointestinal tract at autopsy.
Excessive sweating could be a combination of:

Chronic infection
Diet induced thermogenesis due to eating a lot of meat, which is high in protein, which releases a lot of heat when metabolized (see also "meat sweats"). This same effect can explain why the other man, Charles Domery, who was eating huge amounts of meat, was sweating profusely at night (after eating), despite having no diagnosis of infection, diarrhea or other health condition.

Comments are not for extended discussion; this conversation has been moved to chat.

Tarrare
Tarrare was a showman who was renowned for his insatiable appetite including eating cats, dogs, and snakes sometimes raw.
He is also said to have eaten many inedible items.
At the time, he was alleged to have committed cannibalism of a 14-month-old baby and was caught in the act of cannibalising cadavers by hospital staff.
He was given away as a child by his parents and served in the French Revolutionary Army.
Tarrare died at the age of around 26.
From the book "Anomalies and Curiosities of Medicine" originally published in 1896 by Gould and Pyle:

He was of middle height and was always heated and sweating. He died of a purulent diarrhea, all his intestines and peritoneum being in a suppurating condition.

This record is 100 years after Tarreres death and is largely based on the 1819  London Medical and Physical Journal.
The earlier record claims:

At seventeen years of age, Tarrare weighed only one hundred pounds,
  and was already able to eat, in twenty-four hours, a quarter of a
  bullock of that weight.

From these accounts, I form the opinion that Tarrare ate an incredibly poor diet of raw meat and offal and countless inedible objects.
There are references to throwing up after consuming raw animals and chronic diarrhoea. There are references to sleeping for large periods of time after eating large quantities of food.
Autopsy
A passage from the 1819 London Medical and Physical Journal:

This is the closest article we have to a contemporary record.
From this account we gather that his stomach was enlarged and ulcerated, his gall bladder was enlarged, and his liver was diseased and enlarged.
Also, it might be a factor that his appetite was lost during these final days, common among patients with diarrhoea.
The record at the time, as quoted above, attributes the cause of death to diarrhoea.
Although the picture is painted that the autopsy occurred soon after death, we have no idea if this was hours, days, weeks, and conditions were much less sterile than modern morgues. I am not sure if the  "corruption" mentioned is significant.
One source claims Tarrare died of TB, however, I cannot find any corroborating evidence.
Another source recounts Tarrare blaming his demise on eating a golden fork.
These post mortem observations could have developed from 20 years of eating an exceptionally poor, unhygienic, and often inedible diet and may or may not have been the cause of his underlying condition.
Summary
So we have a few things to consider.

Polyphagia including inedible object consumption
Died young
Gastrointestinal abnormalities identified post mortum
Chronic fever and sweating
Chronic diarrhoea and vomiting
Periods of prolonged sleep

Polyphagia
The London Medical and Physical Journal describes Tarrare as having polyphagia which in his case could not be cured by acids, opium, or tobacco.
Colloquially, polyphagia means ingesting a wide variety of items.
However, if they meant "polyphagia" medically, it could be a symptom.
Polyphagia is a medical symptom defined as excessive hunger.
In modern times, this can indicate a disorder such as diabetes (improper production or reception of insulin) or Kleine–Levin syndrome (a disorder of the hypothalamus).
It could also indicate a genetic condition Prader–Willi syndrome and Bardet–Biedl syndrome.
The full list of causes from Wikipedia is:

Anxiety
Stress
Depression
Orexigenic drugs
Diabetes mellitus
Hyperthyroidism
Hypoglycemia
Fatigue
Premenstrual syndrome
Prader–Willi syndrome

From the
NHS:
Prader-Willi syndrome is a rare genetic condition that causes a wide range of physical symptoms, learning difficulties and behavioural
problems.

Bulimia
Graves' disease

Autoimmune thyroid disorder that can lead to an intolerance of heat

Kleine–Levin syndrome

A sleep disorder

We cannot know if Tarrare's polyphagia was brought on by his stressful exhausting life, his poor diet, or some genetic condition.
We cannot even be sure if his condition was responsible directly or indirectly for his death.
We cannot know if his sweating was related to; his underlying condition, or a chronic state of infection.
The only cause for his eating we can rule out from the above list of underlying causes is premenstrual syndrome (PMS) and orexigenic drugs (which are not documented).
There could be a case made for most of the other causes.
Concluding remarks
Given the scarceness of good quality medical records, the anecdotal nature of what was recorded, and the relatively poor diagnostic power of medical practitioners of the time, it would be, in my non-medical opinion, impossible to conclusively form a diagnosis.

Comments are not for extended discussion; this conversation has been moved to chat.


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Does the Rayleigh formula apply to electron microscopes or only light microscopes?
I am asked to determine the resolving power of both a TEM (transmission electron microscope) and a SEM (scanning electron microscope) and given the Rayleigh formula below.
$$Resolution=\frac{0.61\lambda}{N.A.}$$
Where $\lambda=0.0039$nm since the wavelength of an electron is $0.039$ angstroms and $1Å=0.1$nm.
I am also told that the N.A. the numerical aperture for the TEM is $0.01$
So, $$Resolution=\frac{0.61(0.0039nm)}{0.01}=0.24nm$$
For the SEM I am not given the N.A. but I am told that the resolution is $10$nm
My problem is if I use the Rayleigh formula  $$Resolution=\frac{0.61\lambda}{N.A.}=10nm$$ I get $$N.A.=0.0002379$$ Which would imply that the aperture for the SEM is greater than that of TEM since N.A for TEM is $0.01$ and SEM is $0.00023$ $$0.01>0.00023$$.
My problem is that I'm told the greater the aperture (smaller value of aperture) the greater the resolution but as you can see from the equations above this doesn't seem to make sense, The only explanation I can think of is that the Rayleigh formula does not apply to electron microscopes. Any advice would be greatly appreciated thank you.

This is more than likely better asked in physics SE, however since you contextualized it biologically somewhat I will answer it here.
You are correct in your concern. The Rayleigh Criterion and the associated calculations(Rayleigh Scattering) are talking about the minimum separation of 2 light sources to resolve a given object. From this we are able to derive nice metrics like resolution and so forth. As you say, this describes the resolving of light wrt to the diffraction limit.
In order to calculate the resolving power of an SEM or TEM you need to do a different set of calculations.
First, the theoretical limit of EM microscope resolution is given by the De Broglie Wavelength of the accelerated electrons. However, as with the calculations for the Rayleigh Criterion, there are other factors to consider. For a full walkthrough see this. You take Abbe's equation for resolution in a perfect optical system, and take account of the other factors at play(optics for example).

The resolution of an SEM is not set by the electron wavelength, but rather by the size of the electron beam that is scanned over the sample. Imagine you had a flashlight scanning over a patch of ground, and were recording the reflected light intensity, without using any lenses, to reconstruct the surface features -- your resolution would be roughly the spot size, and not the Rayleigh limit. It's important to keep in mind that the Rayleigh limit is the best resolution one can have; it's easy to do worse!
A TEM, in contrast, does use lensing of electron beams, via magnetic fields.


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Is it mostly true that predators or parasites traverse wider spatial areas than their prey or hosts?
Does it tend to be true that as you go up the food chain, the species tend to cover wider areas?  I am basically asking whether a population's prey varies spatially more than a population's predators and parasites, which would vary more temporally: the predators and parasites to which a population is exposed tends to be the same across the population but the prey vary more spatially because the prey tend to traverse smaller areas.  I would think this were true, since predators have larger body sizes and my thinking is that species with larger body sizes tend to travel farther and parasites may locate on multiple hosts, which collectively carry them farther than any single host.  This is my inference, but I do not even know what to type into Google Scholar in order to check this.

Please show your own attempt at answering the question to avoid having your question downvoted. Thanks.

I edited the question.

Related (but different!): https://biology.stackexchange.com/questions/60627/references-concerning-the-relative-population-sizes-of-predator-and-prey

many prey species have exceeding large migratory routes saying they travel less then predators is unfounded. predators do not always have larger body sizes either.

The ability of prey to find refuge in predator-free habitats is a fundamental hypothesis for predator-prey stability. The availablility of predator-free patches allows the prey metapopulation to maintain positive population growth in some patches, despite being decimated by predators in other patches. If the predators are far superior dispersers, they would eat all the prey, possibly driving the prey—and themselves—extinct.
That being said, it's difficult to make generalizations about spatial food webs. In nature, prey, predator, and parasite populations persist due to a multitude of factors, including the spatial configuration of suitable habitats, their dispersal rates, interactions with competitors and mutualists, etc. For example, in a system where carnivores hunt herbivores, sure, the predator may disperse across the landscape, hunting across more than one herbivore metapopulation; in other systems, such as those that may be more spatiotemporally variable (e.g., prone to disturbance and local extinctions), the ability of the prey to disperse faster than the predator could be a crucial aspect of predator-prey stability.
Some theoretical resources include: Comins et al. 1992, Holt 2002,
Amarasekare 2008.
A foundational experiment is that of Huffaker, who dispersed mites on oranges: Huffaker 1958. Also, take a look at work on host-phage interactions: Schrag and Mittler 1996.
For Google Scholar searches, try various combinations of the following words: spatial, host, parasite, predator, prey, stability, coexistence, food web.

Yes there is a bit of correlation for animal weight, it's a very vague graph. No, food chains don't correlate predictably well because of the different locomotion energies of birds, mammals, insects, wind transport, river and tide transport, and ecosystem differences.
Every time there is a different locomotion type and energy, it makes exceptions. flying animals like geese, sparrows and locusts can travel thousands of miles compared to their all of the predators, eagles, foxes and spiders.
It's too general. It's 50 questions in one, you encompass millions of species. There isn't a clear study to consider for it. Parasites places in the food chain is not very clear, it's another topic too. Parasites are the contrary, they are small, so the host can travel more than the parasite, even if it's attached like a tick.
Elephant home range varies from 50 to 3000km,
here are home ranges, which can vary 10,000 times for the same wieght animals:


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How would you model the evolution of two genotypes across generations?
Say you have a genotype A that produces x offspring and another genotype B that produces y offspring, where x>y.  These x offspring are of genotype A but with modest differences in fitness due to mutation and these y offspring are of genotype B but with modest differences in fitness due to mutation.  How would you model how many offspring these x offspring can themselves produce and how many offspring these y offspring can themselves produce as a function of the quantity their original parents produced?
Obviously, you could say that each descendant of genotype A produces x and each descendant of genotype B produces y, but that would unrealistically benefit genotype A, since realistically, one of B’s offspring could have a chance to be fitter than A’s by developing a favorable mutation or one of A’s offspring could have a chance to be less fit than B’s by developing an unfavorable mutation.
You could say that each descendant of genotype A and B produce (x+y)/2, but this would not be fair to genotype A, since it is fitter, so its offspring would probably be fitter than B’s offspring.
You could use the breeder’s equation and say that each of A’s offspring produce xh+m(1-h), where h is the heritability of fitness and m is the population mean, and that each of B’s offspring produce yh+m(1-h).  But this is also unfair to genotype B because all of its offspring are less fit than all of A’s offspring, since x>y.  So, what’s a way of modeling how resultant mutations might occur, such that B has a chance of producing some offspring that are fitter than A’s offspring?

A few questions: 1) you seem to be conflating fitness and number of offspring. How sure are you that this is a good idea, if only on a conceptual level? 2) "m" is population mean of what, fitness=number of offspring? Does this mean "m" changes as the makeup of the population changes, or is it just the mean of "x" and "y"? 3) if "h" is nil then A's offspring produce "x" offspring and if it's 1 they produce "m"; shouldn't it be the other way around?

You seem to ignore the variation you postulated within your populations. It all depends on that; for example if the 2nd-gen fitnesses are normally distributed with a mean of the parent gen fitness, then "but what about B's offspring that are fitter than A's" doesn't necessarily matter, since B's less-fit offspring would be less fit than A's least fit, and A's fitter offspring would be fitter than B's fittest (i.e it will come down to the mean, which is x and y). And if the distributions between the two are different then all bets are off. Surely this needs to be in the model somewhere.

(for example you said each descendant of A producing x and each descendant of B producing y unfairly benefits A, but if the variation in fitness is that half the offspring produce "parent fitness + z" and half produce "parent fitness - z", then do the calculation you'll find it's equal to if each descendant of A produced x and each descendant of B produced y).

Thank you for the questions.  1)  My understanding of fitness is that it has a number of definitions depending on context and I think my usage of the term is okay.  Fitness covaries with the ability to produce offspring.  2)  Yes, m is population mean of fitness (or number of offspring).  m should change as the makeup of the population changes.  I am not suggesting I should use heritability in this way though.  Just gave an example of what I'm trying to get at.  3)  You're right.  I apologize.  Will edit it.

A's fittest offspring wouldn't necessarily be fitter than B's fittest.  It would just be more likely.  B's less fit offspring would not necessarily be less fit than A's less fit (especially since A would have more offspring).  And yes, that would also be unfair to B because it wouldn't give B the possibility of having more grandoffspring or greatgrandoffspring like some kind of random process would.

"A's fittest offspring wouldn't necessarily be fitter than B's fittest. It would just be more likely." But that assertion depends 100% on how your fitness varies from parent to offspring. That variation must follow an equation (a probabilistic one if needed); if you've decided what it is then it should be in the model. If you haven't decided what it is then you need to decide, because that equation will determine your results more than anything. I already gave an example of one (clearly not what you have in mind) that would give results contradictory to what you seem to expect.

What I'm asking is what is a good way of representing this?  That is, what is fair to both A and B?  Have you seen anything in the theoretical literature that models something like this?  You gave a good example, but in that example, B would have no shot of having more grandoffspring.  I haven't seen in which offspring fitness varies as a function of a parent's fitness.  The assumption commonly made is that the fitnesses of all offspring are equal, which is unrealistic.

Sorry, I hadn't seen (or had forgotten by the time I wrote my later comments) that "what equation would allow B to have fitter offspring than A" is basically your question. I'll put the rest in an answer.

Allows B to have fitter offspring than A in a realistic way.  Obviously, A would be more likely to have fitter offspring, but B would have an opportunity.  How has/can this be represented?  Thank you.  I look forward to your answer.

So, what’s a way of modeling how resultant mutations might occur, such that B has a chance of producing some offspring that are fitter than A’s offspring?

Here's one possible solution that fits your criteria. You could have something like this (with "d" being the distance between "x" and "y", i.e. d=x-y and "n" being the n-th offspring, so it varies between 1 and x for A and 1 and y for B) :
A: n -> x + r(n), where r(n) is a random number between x-d and x+d
B: n -> y + r(n), where r(n) is a random number between y-d and y+d
(I write r(n) not because r is a function n, but because a different number is drawn for each n. I also didn't specify r's distribution, that would also need to be decided when making an actual model)
This is your situation where some offspring of B have a chance of being fitter than offspring of A, and the least-fit offspring of B can even be fitter than the fittest offspring of A. I think it fits your criteria and thus answers your question. The sum of the grandoffspring is basically the basic x or y squared (that you would get if each offspring had the same fitness as their parent) + the sum of all the "r"s.

OK thank you for the effort you made.  Can I upvote your answer but not give it a check because I would like to leave the question open for other ways of modeling this (perhaps ways people have seen in the literature)?  Can there be multiple "accepted" answers in a thread?


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What is it called when there is natural truncation selection?
Truncation selection is when a breeder selects for animals that exhibit a value for a trait that is above a certain threshold.  What is it called when nature selects for a value of a trait that is above a certain threshold?  I want to say natural truncation selection, but I don't see any mention of that.  To clarify, what I mean is, what is it called when, as a result of natural selection, all perish but those with a value for a particular trait that exceeds a particular threshold.

The term that comes to my mind is directional selection, but I'm not sure that's what you're asking about?

Truncation selection
The term  truncation selection has been used a lot in theory papers because it is a simple model and because it is of interest to breeders. Truncation selection is most likely more common in a breeding setting than in nature and from wikipedia,  truncation selection is defined as specific to breeding

In animal and plant breeding, truncation selection is a standard method in selective breeding in selecting animals to be bred for the next generation

However, as much as the definition fail to repeat animal or plant instead of just animal, IMO it fails to recognize that such notion could as well exist in nature. I would personally not be shock to read a paper talking about  truncation selection in wild populations.
Threshold model
If you really want to avoid talking about truncation selection, then you can eventually picture the phenotypic trait as a liability factor (as it is done in sex determination literature, see Perrin and Beukeboom's book) and selection acting as a threshold on this factor. Note that the concept of threshold and threshold model is very common and everyone will understand what you mean.

"If you really want to avoid talking about truncation selection, then you can eventually picture the phenotypic trait as a liability factor (and it is done in sex determination literature, see Perrin and Beukeboom's book) and selection acting as a threshold on this factor." Can you expound on what you mean by this?

In the sex determination literature, sex is often considered as a threshold trait on some liability factor. For example in humans, SRY create TDF and TDF is the liability factor. If TDF is at a high enough concentration, it will be a male, otherwise a female. Similarly, you can picture fitness as a threshold trait where your continuous phenotype of interest (heigh or whatever) is the liability factor.

Would you define "liability factor" please?

I won't be able to cite you a nice neat definition, but I mainly encountered this term in the sex determination literature and it is some factor (protein concentration typically) which depending on its value will affect a phenotypic trait based on a threshold model. I am not sure the term is always used like this though. Sorry, I can't give you more info on that. If interested you might want to have a look at this book.


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Can cooking oil kill fleas?
I cannot find a reliable way to kill fleas after removing them from my cats with a flea comb.  Soap and water does not do the job and they climb back out of the drain.
Will cooking oil  suffocate fleas?  If so how long of time until the flea expires?

You can easily drown them in a glass of plain water; they stop moving within 10-20 seconds, but you could leave them longer to make sure. Just don't look closely at them during this, as some may find it upsetting.

Short answer: Mire them in a viscous liquid then leave them in there long enough for them to die, instead of immediate disposal.
Cooking oil is viscous, so yes you can viably use it to mire the fleas. You'll need to wait until they die though to dispose of them or else they'll come crawling back as you've stated. The DIY option is generally a water/dish soap mixture in a container: you'll put the fleas in and they sink to the bottom almost immediately. Just leave them in there and they'll eventually die in about 5 minutes. Some individuals suggest Dawn dish soap, much like here, where the claim is that the Dawn contains compounds which suffocate the fleas.
You can also make a flea trap for around the house, with a bowl of dish soap and water near a night light. They'll jump for the light and end up falling into the flea trap.


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RNA-Polymerase III transcribed genes
I have a list of differentially regulated genes from a high-throughput experiment of the human genome. I would like to find out which of these genes are transcribed by a RNA polymerase III and which of them (probably most) are transcribed by the RNA polymerase II.
I have downloaded some Chip-Seq tracks for polymerase II and III and some GO categories which apply for the RNA polymerase III transcription.
I have also tried the biomaRt repository to search for these mentioned GO categories, but I can't really find substantial results.
For example I have the gene RN7SL1 which according to the literature is transcribed by PolIII, but I'm not able to find any repository which also says so.
Is there any (better) way to search for this kind of data/annotations?
thanks a lot
Assa
PS.
I am not sure, if I have found the correct GO categories (e.g. GO:0006383 - transcription from RNA polymerase III promoter). So are there any better one?

GO is based on experimental evidences; biomart is quite good. GO:0006383 seems to be the right tag.
If you want to use the ChIPseq data then you can do this:

Map the ChIPseq reads to the genome. If you already have the tracks
then you need not do the mapping. You would have the position of the
reads in the genome.
Now count the number of reads mapping to each geneic region. The gene boundaries can be obtained from a GTF file.
Optional: Normalize the reads with the length of the gene.
Find the difference between the number of Pol-II and Pol-III reads for each gene.

By doing this you still won't be able to say that "gene-x is transcribed by pol-III". All you can say is that "pol-III binds to that geneic region". If there is an even distribution of the ChIPseq reads along the gene then you can assume that there is an active transcription and the polymerase is not merely stalled. You can also do a CLIP (Crosslink-Immunoprecipitation) of Pol-III followed by a RNAseq. This will enable you to find RNAs transcribed by pol-III (this is a better way than ChIPseq but I don't think anyone has done it yet; so no ready data).


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How can I reduce the background gradient in phase contrast microscopy?
I'm looking for ways to set up a phase contrast microscope to reduce the brightness gradient that appears across the background of my images.
I use phase contrast to find the outlines of cells in my images. My problem is that the automated processing algorithms I use get confused by strong brightness gradients in some images. Post-processing of the images disrupts the continuous nature of the histogram, again throwing off the algorithms.
Does anyone know why these gradients appear and how to reduce their formation?
Thanks,
Oliver
Edited the question to clarify that I am interested in microscope usage, rather than image manipulation
Sample image:

The image background is darker at the left of the image than at the right

Are you using the images to count cells or to determine shape, volume etc? Also, the question is borderline off-topic since it is mostly about image manipulation, so it would probably be good if you added some more info on the biological background/problem you are studying.

@fileunderwater I use the phase image to create ROIs for collecting data from epifluorescent images. How I use the images is not particularly important with respect to the question, though. I'm just interested to know if there are ways to configure my microscope such that the brightness gradient across the field is less pronounced.

Add this information directly to the question by editing. For the record, I think the question is fine since its an important lab techique (but I cannot answer it), but it might attract close-votes for being "only" about microscopy and not biology.

I hope not - there isn't a microscopy stack exchange I can post in instead! The biology exchange does contain many other questions purely related to microscopy, though... Furthermore, I expect biologists to comprise the largest demographic of microscope users, so I'm more likely to find an answer here than anywhere else. Thanks for your comments, I've edited the question accordingly.

Is it possible to provide an image of the problem?

I've edited the question to include a link to an image.

What's the microscope your using? Have you tried adjusting the condenser??? Also check your apertures.

It can be because of a silly error of a partially closed lens (the knob that lets you switch from camera to binoculars). That happened to me once.

Have you tried imaging an emptry slide and use it as background? (i.e. subtracting it from your image)

That's a good idea, @nico - I'll try it next time.

@WYSIWYG (and rhill), I have a similar problem sometimes where the condenser on our leica scopes can slide and doesn't always fully engage, but that's not so subtle (it completely disrupts the phase contrast effect). If anyone reading this is having trouble getting phase to work, the phase condenser is a fairly common cause.

If you're not already doing so, make sure you set up Kohler illumination every time you use the microscope -- see this tutorial by Steven Ruzin.
The lamp or the phase ring may be misaligned. There are usually small hexagonal set screws on the lamp housing and the condenser, respectively, to adjust these.
Imaging near the edge of a dish or well can lead to uneven illumination as the light is bent by the walls of the culture vessel. If this might be the cause, try re-doing the Kohler illumination before you take each image.
If you can't find the cause of the problem, there are several ways to correct for uneven background described here. Maybe one of them will work with your analysis.

Excellent first post! Welcome to Biology S.E.! If you need any assistance, please visit The Help Center.


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How does the size of insert affects the rate of Homologous Recombination in yeast?
When performing genetic knockouts in yeast using homologous recombination to replace a target gene sequence via a vector DNA, does the region between the flanking regions in the vector have to be the same length as the site of recombination in the host cell's gene?

If I understand correctly, I think what Adelina's asking is if the DNA between the flanking regions (i.e. the insert being integrated) needs to be the same length as the gene being knocked out. The answer is NO.

As pointed out by aandreev, the greater the size of the insert relative to the flanks lower will be the recombination rate. This is because the donor DNA essentially becomes non-homologous. See the figures below:

Figure1: Effect of insert size on recombination efficiency. Kung et al. (2013) Appl Environ Microbiol. Mar;79(5):1712-7

Figure2: Effect of flank size on recombination efficiency. Kung et al. (2013) Appl Environ Microbiol. Mar;79(5):1712-7

However, you mentioned that you want to make gene knockouts. In this case you just need to make a small mutation (for example introducing a premature stop codon).
Use of genome editing tools such as Crispr-Cas, ZFN and TALEN increase the rate of transgenesis by introducing DNA double strand break.
These can also be used for making knockouts when NHEJ dominates over HR as the DNA repair mechanism. NHEJ is error-prone and can cause indels that can disrupt gene function.

Impressive. Nicely researched work. +1

first figure is very nice. Also, I will use all of them to argue in the lab later.

In my view there is direct link between probability of recombination and size of the insert. Smaller inserts with larger flanks get incorporated easier. That I understand from reviewing studies based on Cas9/CRISPRs system. Same should stand for HR-mutagenesis in yeast.
So, short answer will be: shorter inserts are more efficient. However, I couldn't find any studies of relation between recombination efficiency and using smaller than target inserts, or relation between sizes of target and insert. Cas9/CRISPR system was used to insert loxP site (34nt) instead of ~10 bp in mice ES cells. People usually try to insert GFP or something similar in size instead of shorter genomic sequences.

authors: Иван Бондарев


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Minor Allele Frequency (MAF) higher than 0.5?
I have a set of data in PLINK format (*.bed, *.bim and *.fam files).  There are around 2000 individuals in my dataset.
I used PLINK to calculate allele frequencies in the population:
p-link --bfile data --freq

However, in the produced results file, many SNPs have a MAF over 0.5 (up to 0.82).  Would that not then make that the major allele frequency, as the other allele would have a frequency of 0.18?

can you add some more info about PLINK format and background info for the question so other users not as familiar can follow?

You should post an example section from your data file that reports MAF>0.5.