How do scientists know what part of DNA does what? There are many genetically engineered foods and animals and I was wondering, how do scientists know which part of a DNA to cut in order to produce the desired result. For example I want to make a tomato more durable and to do that I cut the durability part of the DNA of something long lasting and put in the tomato. I'm not sure, but I guess that's not how things work. If I wanted to make a human with tiger arms, I'll just cut the arms DNA part of the tiger and put it in the human DNA, no? I'm sure it's much much more complicated than that.

P.S - I'm just 16 and interested in biology, I do not posses any university/professional academic knowledge.

This is a tough question (scientifically)! Generally speaking, it's impossible to look at a DNA sequence and predict what function it might have. It's easier to go backwards: identify a function/trait of interest, figure out what proteins are necessary for that function, and then deduce the sequence.

I'll describe one strategy to go from protein to gene, and name some techniques which you can read up on if you want to learn more.

To find the amino acid sequence of a protein, you can chop it up using proteolytic enzymes. This gives you fragments of varying lengths, since the enzymes will cut semi-randomly at different sites in the protein. Run these fragments on chromatographic paper. You can deduce the amino acid sequence from the pattern, because larger fragments will move slower. This is the same idea behind Sanger sequencing (aka chain termination sequencing) of DNA, and was invented by the same guy.

If you know what the amino acid sequence is, you now know the mRNA sequence, since protein is translated from mRNA. However, in eukaryotes, you don't know the DNA sequence, since we have introns - i.e. bits of the sequence which get cut out between transcription and translation. We need a way to get from mRNA (protein precursor) to DNA (gene).

To do this, purify mRNA from a cell and generate a library of complementary DNA (cDNA) using reverse transcriptase. Then, sequence the cDNA library to generate expressed sequence tags (ESTs), which are subsets of the full cDNA sequence. Because the ESTs are subsets of the sequence instead of the full transcript, it's possible to "CTRL+F" the organism's genome and find the gene which contains the EST.

Scientists have been doing this for decades, so nowadays you can search a gene/protein database and instantly know which sequence is responsible for, say, a specific channel protein.

To go in the other direction from gene to protein to function/trait, you have to do a fair bit of legwork as well. In very, very simplistic terms, you "cut" some DNA, put it into a package, and deliver the package to your favorite organism. As you said, it's a bit more complicated than that. :)

For complex, polygenic traits, it's harder to (1) identify the genes responsible and (2) successfully engineer an organism to have all of those new genes. You're unlikely to see a human with tiger legs any time soon. It's much easier to give an organism a few genes that confer very specific traits. Look at this list of genetic modifications to crops and click on one of the traits; usually only 1-3 genes have been introduced.


Why is electroporation not the prefered choice transforming for Mammalian Cells (HEK)? I see a lot of folks using different techniques for transforming mammalian cell (specifically HEK) instead of doing electroporation like I see with E.Coli (bacteria). Is there a reason for this ?

Viral methods are just more efficient and give you a higher viability in the end. Electroporation works but you'll have a low yield of viable cells after. Here's a nice paper looking at the effects of buffer composition on cell viability/transfection efficiency: https://www.nature.com/articles/s41598-020-59790-x with the general take away being, if electroporation is working well enough to transfect the cells, it will also kill a fair percentage as well.
Hek cells in particular are pretty easy to transfect through viral methods for stable integration of your plasmid or Fugene/lipofectamine for transient expression.


Effect of testosterone hormone on cell signalling and behaviour? A steroid, testosterone was injected in female body which led to development of secondary sexual characters but these characters were not developed over night (Response was very slow). What could be possible explanation of this phenomenon

a Steroid hormones produce a slow response because they do not have secondary messenger
Slow response is due to lack of cell membrane receptors and amplification enzymes
Movement of this hormone is very slow in blood
None of the above

I think option 3 is correct because steroid hormones are hydrophobic in nature and cant be transported in the blood without the help of any carrier protein so this may be the reason but I am not sure about this..

Steroid hormones produce a slow response because they do not have secondary messenger
It's true that steroids don't have secondary messengers because they don't need them - they can go directly into a cells nucleus and affect transcription there.  This wouldn't slow down the response by much, however.
Slow response is due to lack of cell membrane receptors and amplification enzymes
This is just the inverse of the first option, which again isn't really a cause of slowing down the response.  Steroids are amplified in that they act directly on DNA at the very first step of the synthesis pathways so don't need much amplification.
Movement of this hormone is very slow in blood
 Again I don't think there is much to this option, the difference in speed of distribution of steroid and peptide hormones is likely to be minimal

This leaves us with:
 4. None of the above

I would personally have gone with this answer.  Steroids have a long time of action because they require the new transcription of genes that they activate.  This process takes time and consistent doses are needed to maintain the effect.


What is an isolated beta bridge? DSSP gives the letter B for a "residue in isolated β-bridge (single pair β-sheet hydrogen bond formation)", according to Wikipedias page for secondary structure (and various other proper sources). Can somebody please explain what this is in some more detail please? If I google it all that comes up is pretty much the same as what wikipedia gives.

In Kabash and Sander's paper related to DSSP (Biopolymers 1983 vol. 22 (12) pp. 2577-637) the following appears in the abstract:

We have developed a set of simple and physically motivated criteria for secondary structure, programmed as a pattern-recognition process of hydrogen-bonded and geometrical features extracted from x-ray coordinates. Cooperative secondary structure is recognized as repeats of the elementary hydrogen-bonding patterns “turn” and “bridge.” Repeating turns are “helices,” repeating bridges are “ladders,” connected ladders are “sheets.”

It seems from this that they are using the term bridge to mean one hydrogen bond of the type found in a beta-sheet, ladder as two protein strands joined by many hydrogen bonds, and sheets as beta-sheets — more than two protein strands joined by many hydrogen bonds. The term beta-bridge in the DSSP documentation (http://www.cmbi.ru.nl/dssp.html) is presumably equivalent to a bridge in the paper

In the DSSP files residues that are in ladders or beta-sheets are designated E, and B is used for instances where there is a single hydrogen bond. The former are very common, wheres the latter are quite rare — some proteins have none. I have prepared an image of an example for residues 74 and 79 (side-chains omitted for clarity) in a loop in the protein 1PPR.pdb (a light-harvesting protein). The other loop residues 75-78 are designated as T (turn) in the DSSP file, whereas residues 73 and 78 have no structure designation. Hence the hydrogen bond between Ser-74 and Val-79 is an isolated singleton and designated B.


Why was Darwin's Finch argument valid considering Weismann's argument? According to Darwin, there was only 1 species of Finch bird in Galapagos. Then Darwin argued that due to diet adaptation and natural selection, their beak changes causing new species--which is confirmed by John Gauld at the Geological Society of London.

In contrast, Weismann's and his mouse proofed that environment factor won't effect next generations.

Don't Weismann's argument contradicts Darwin? Ok, say that the Finch bird adapts (and killing the one who don't) with their diet. Their beak changes. But when these Finch bird breeds, their new generation's beak should reset to normal beak again.

The reason I asked this reason is because one article said that our teeth are changing due to overbites using utensils(1). However, not everyone use eating utensils. Indian mostly used finger. Indonesian don't really care whether we use our hand or spoon. If this trend keeps happening in the future, won't human evolve into two species? Homo sapien and Homo spoon (pun intended.)

Or am I mixing Darwin with Lamarck?

(1):http://www.theatlantic.com/health/archive/2013/01/how-forks-gave-us-overbites-and-pots-saved-the-toothless/267252/

Weismann's mouse tail experiment showed that mouse tail length is not inherited, if the change in tail length was caused by mutilations, and there was an absence of selection pressure.

However, Darwin's finches represented a completely different idea of evolution. In this case, the beak size of the finches did in fact change, but this was not due to mutilation. The beak sizes of the finches changed because finches with lower fitness due to their beak sizes were selected against, leading to the beak size changing over time.

If the mice were instead selected for tail length by preventing the long-tailed mice from reproducing, then it is indeed possible that the mice would eventually evolve to have shorter tails, or even absent tails.

Similarly, if the finches had their beak sizes reduced artificially but their fitness preserved (for example, by grinding down their beaks and feeding them soft food that does not require large beaks to break), the finches would not be expected to evolve smaller beaks.

It is not clear which method is the one through which the evolution of the overbite occurred. It could be due to a change in relative fitness, but a case can also be made in which fitness was not a large factor.


Assumptions for Nernst Equation While I am familiar with some of the conditions for the Nernst Equation, for example:

1) "The membrane is only permeable to one ion even if there are several other ions in the system"

there is an assumption/condition that I have yet to see mentioned (and I am pretty sure that it is an essential one that must be included). That assumption is as follows:

The two solutions separated by the selectively permeable membrane must initially have the same total charge...i.e. there is no initial potential difference between the 2 sides of the membrane

I believe this must be true because of the following 3 cases:

for all 3 cases, only potassium is permeable, and there is a "Left Side" and a "Right Side" on either side of the selectively permeable membrane.

A) Left Side: 25 mM K+, 125 mM Na+ |&| Right Side: 125 mM K+, 25 mM Na+

B) Left Side: 25 mM K+, 10000000000 mM Na+ |&| Right Side: 125 mM K+, 25 mM Na+

C) Left Side: 25 mM K+, 0.00000000001 mM Na+ |&| Right Side: 125 mM K+, 25 mM Na+

Without using my proposed assumption, in all 3 cases, the Nernst equation for potassium would be the same:

(RT)/(ZF) * ln (25/125)

However, I fail to see how case B & C could have the same Nernst potential for potassium as case A when the electrical forces on the left side of the membrane are completely different! Let's use case B as an example. Clearly, the potassium concentration differences exist between the two sides (25 mM vs 125 mM), BUT there is an enormous electrical repulsive force imposed by the sodium (1000000000 mM Na+) so there is no way that the same amount of potassium will pass over to the left side as it would in Case A, right?

Could someone please clarify?

Assumption (1) is not really even true. The Nernst equation is always valid to find the reversal potential for a single ion: that is its purpose. To determine the equilibrium potential for a number of ions, you use the Goldman/GHK equation. It happens to be that the Goldman equation reduces to the Nernst in case of single-ion permeability, which of course never happens for real but is a reasonable approximation in some cases.

As far as your additional assumption, no, that assumption is not required. The Nernst equation doesn't tell you which directions ions flow, it tells you what the reversal potential is for an ion. If you add a voltage from some other source (like your example of adding a bunch of mysterious unaccounted for positive charges outside the membrane) that doesn't change the reversal potential, it changes the voltage. If you put a large positive charge on the left side somewhat like your situation (B), potassium would flow up its concentration gradient towards the Nernst potential for potassium.

Note also that your example is utterly ridiculous: if you want to know what happens when you have 10000000 M Na+ ions together on one side of a membrane you need to consult a physicist, not a biologist. I don't know where you put all the other negative charges, but you're probably about to have a) A very expensive electric bill, and b) a very big hole in your lab.

In real biology, the number of charges on each side of the membrane is similar enough that you can call it equal (see for example Why is it possible to calculate the equilibrium potential of an ion using the Nernst equation from empirical measurements in the cell at rest?). In any case where someone describes "adding sodium" to one side of the membrane, they don't mean just sodium, they mean sodium as a salt with some negative ion, it's just that the identity of that negative ion doesn't matter if it isn't membrane permeable (relative to the other ions involved).


Exponential growth equation and bacteria I'm a microbiologist, but I'm teaching some ecology in my intro-level course, so when we got to population growth, I thought I'd use the example of a microbial population. But, I hit a strange problem that I thought maybe an ecologist could help me understand:

Imagine a population of bacteria that can divide every 30 minutes. In one hour, every cell produces four cells. Since no one really dies, the intrinsic growth rate (r) is 4. The exponential growth equation, dN/dt = rN works fine to show the growth of the population: starting with one cell, in one hour it's 4, then in two hours rN = 4*4 = 16, in three hours rN = 16*4 = 64 and so on. At 16 hours, we get to about 4 billion bacteria, which is exactly what the microbiologist expects.

But, we then tell the students that to make it easy to predict future numbers, we can do some math and get N(t) = N(0) e^rt. If N(0) = 1, t = 16 hours and r = 4 bacteria/hour/cell, this gives e^64 which is about 6x10^27. Yikes! Kinda far from 4 billion.

So...why doesn't this work? I feel like either I'm not understanding r correctly (it would have to be down around 0.6 for this to work out, I think) or maybe there's a limit on the equation and it just doesn't work for the absurdly high r of bacteria?

Thanks for the help...

Considering your assumption:

  I'm just looking at the exponential part, where the simple exponential
  equation works. If we assume there's sufficient nutrients for bacteria
  to grow unchecked for a number of hours (more-or-less true in a real
  culture)

In your original model you are using discrete states and fixed time steps. So, if 30 min is one time step then after n-steps you have $2^n$ or $4^{(n/2)}$ cells. You are basically counting double steps, which is fine. For a continuous approximation you should estimate the rate like this.

You have the first-order growth equation:

$$\frac{dN}{dt}=rN$$

When you integrate this equation (definite integral) you'll get:

$$ln\left(\frac{N_f}{N_i}\right)=r\tau$$

Where $N_f$ is final number of cells, $N_i$ is initial number of cells and $\tau$ is the time interval.

When the cell number doubles then $\dfrac{N_f}{N_i}=2$ and $\tau=30\ \text{min}$ (doubling time).

 Then your rate constant would be: $\dfrac{ln(2)}{30}\approx0.023\text{ min}^{-1}$

After 16 hours (16×30min) your number of cells would be: $e^{0.023\times16\times60}\approx 4.27\times10^9$ (approx 4 billion).

So what is basically wrong? You haven't rescaled your rate. Since the exponent is $e$ and you know that $a^x=e^{x.ln(a)}$, all you need to do is scale the rate constant with $ln(a)$ (a=2). Moroever, in a continuous model, you don't have fixed time steps. So you scale the constant with the doubling time as well.


Trimming of tRNA precursors From Molecular Biology of the Cell (4th edition) by Bruce Alberts et al. (Chp 6, Pg 338) :

  Both bacterial and eucaryotic tRNAs are typically synthesized as
  larger precursor tRNAs, and these are then trimmed to produce the
  mature tRNA. In addition, some tRNA precursors (from both bacteria and
  eucaryotes) contain introns that must be spliced out.

What's the difference between the two statements (and between trimming and splicing) ?

Trimming = removing RNA sequences from one end.

Splicing = removing introns and joining exons back together.


Will the "frog in boiling water not jumping out" work on warm blooded animals There is the famous saying about a frog that is put in water that are slowly boiling will not jump out until it's too late. I realize it happens because of the frog's cold blood that adjusts to the temperature change until it's too late.
Will this work as well on an animal with warm blood, or will the animal jump out ones it gets hot, but not hot enough to cause permanent damage?

Edit due to comments:

Cold blooded animals, like the frog, can adjust easier to their surrounding when the surrounding changes temperature gradually as their body temperature adjusts with the surroundings. Will the same happen to a warm bodied creature, and will it find it easier to adjust to the surrounding if the change is gradual.

For example, I don't like saunas, when I get in them I have problem breathing and I can't stand the heat, which means that I can be in a sauna for a very short time until I have had enough and get out. Will putting me in a sauna at room temperature and then turning it on to gradually begin to heat until its destination temperature will allow me to fill more comfortable and stay longer inside once it's at the normal sauna temperature?

This answer belongs on Skeptics. Sorry to disappoint, but the "boiled frog" phenomenon is an "old folk warning". This essentially negates your original question altogether. Neither a cold-blooded animal (such as a frog) or warm-blooded animal will boil to death under the conditions implied by the warning (i.e. escape is permitted and water is heated very gradually).

1897 research by German scientist E. W. Scripture, upon which the fable may have first been based, has been deemed flawed by scientists:

  From Scripture's research: “. . . a live frog can actually be boiled without a
  movement if the water is heated slowly enough; in one experiment, the
  temperature was raised at the rate of 0.002 degrees Celsius per
  second, and the frog was found dead at the end of 2.5 hours without
  having moved."

  According to Dr. Karl S. Kruszelnicki (Australian scientist): "[T]he numbers just don’t seem right. If the water comes to a boil,
  that means a final temperature of 100 degrees Celsius. In that case,
  the frog would have to have been put into the water at 82 degrees
  Celsius. Surely, the frog would have died immediately."

  According to Dr. Victor H. Hutchinson (Herpetologist and Zoology Professor at University of Oklahoma):
  "The legend is entirely incorrect! The 'critical thermal maxima' of many species of frogs have been
  determined by several investigators. In this procedure, the water in
  which a frog is submerged is heated gradually at about 2 degrees
  Fahrenheit per minute.  As the temperature of the water is gradually
  increased, the frog will eventually become more and more active in
  attempts to escape the heated water.  If the container size and
  opening allow the frog to jump out, it will do so."

Whit Gibbons (University of Georgia) says that there is an important message behind the false legend:

  So where does that leave us with the boiling frog as a metaphor for
  the human response to economic change or environmental degradation?
  Well, it's not true that you can induce a frog to willingly remain in
  boiling water by starting it off in cold water. But that does not
  diminish the truth of the message that the accumulation of
  imperceptible changes can have a significant effect on the economy and
  the environment. We need to be aware of what changes are occurring and
  to respond to them in a timely fashion. The metaphor lies in the
  frog's ability to escape from the container: if there's no way out,
  then the frog's fate is a foregone conclusion.


Mechanism of redirected blood I've read several fitness articles mentioning redirected blood flow to muscles that are in use. However, they never mention how it works. Is it local and specific to the muscles in use? Or, for example, I guess more blood has to flow through the entire arm if I am training my forearms..? So, won't the entire arm benefit to a certain extent?

I did some research and discovered the vascular shunt mechanism through the opening and closing of capillary sphincters. However, I cannot find any resources discussing how local this effect is. So, as an example, how much does blood flow to the arm improve if someone is training their forearm for six months?

A major determiner of blood flow redirection among muscles is increased oxygen demand in the working muscles.

When a muscle works, it uses oxygen, so the partial pressure of oxygen (PO2) in the muscle falls, which signals small arteries (arterioles) to dilate, which results in increased blood flow and hence oxygen and nutrients delivery to the muscle (Skeletal Muscle Circulation, NCBI Bookshelf).

When you exercise your forearm muscles without exercising upper arm muscles, the arterioles in the forearm muscles will dilate but in the upper arm muscles will not. The blood will just flow through the large arteries of the upper arm toward the forearm, but it won't be delivered to the upper arm muscles.

A long-term effect of exercise is the formation of new capillaries in the exercised, but not in other muscles (PubMed, 2008). So, six months of exercising forearm muscles without exercising upper arm muscles will result in better circulation only in the forearm muscles.


Why does spraying water on plants help them survive in case of frost? I've heard that spraying water on plants will help them survive the frost. Why is that?

Some say that you should do it in the morning right before the sun rises, and others spray water the whole time of low temperatures.

What is the explanation for why this spraying techniques works?

The question has already been asked in Physics.

According to the information there and here the sprinkling must continue throughout the period when temperatures are below freezing, and is only effective for temperatures down to around -5 °C.

Explanations invoke three interrelated effects: when water freezes heat is released so there is a warming effect; the temperature of an ice-water mixture is 0 °C; and an ice layer will insulate the leaf. As long as the temperature of the leaf stays above freezing there will be no frost damage.


Why clotting does not occur during Menstruation As we know when we get injured our body's clotting systems stop the bleeding. So why does it take days for menstruation to stop ?

The endometrium changes throughout the menstrual cycle in response to hormones. During the first part of the cycle, the hormone estrogen is made by the ovaries. Estrogen causes the lining to grow and thicken to prepare the uterus for pregnancy. In the middle of the cycle, an egg is released from one of the ovaries (ovulation). Following ovulation, levels of another hormone called progesterone begin to increase. Progesterone prepares the endometrium to receive and nourish a fertilized egg. If pregnancy does not occur, estrogen and progesterone levels decrease. The decrease in progesterone triggers menstruation (the shedding of the uterine lining). Once the lining is completely shed, a new menstrual cycle begins. (ACOG)

The uterine lining secretes an anti-clotting factor during periods to assist the blood flow. If the substance is not sufficient to overcome the natural clotting factor menstrual blood clots can form, a process known as hemostasis. Flow may slow down toward the end of a period, and blood can clump. When women are reclining for a time, such as while sleeping, the discharge will pool, and blood clots will be dislodged when upon standing. The blood darkens as it sits and sometimes looks brown or black.
During menstruation, the endometrial cells that line the uterus strip away and leave the body. As this happens, the body releases proteins that cause the blood in the uterus to coagulate. This coagulation prevents the blood vessels in the uterine lining from continuing to bleed. The blood that the body has already shed also contains these coagulation proteins. When the flow is most substantial, the coagulation proteins within the blood may start to clump together, resulting in menstrual clots. This generally occurs when menstrual blood pools in the uterus or vagina before leaving the body.
Menstrual blood clots can be caused by an imbalance in the clotting and anti-coagulating factors in blood or by hormonal changes. Fibroids and endometriosis contribute to excess buildup of the uterine lining, which breaks free as clots.

Under normal physiological circumstances menstruation is a highly regulated, complex process that is under strict hormonal control. During normal menstruation, progesterone withdrawal initiates menstruation. The cessation of menstrual bleeding is achieved by endometrial haemostasis via platelet aggregation, fibrin deposition and thrombus formation. Local endocrine, immunological and haemostatic factors interact at a molecular level to control endometrial haemostasis. Tissue factor and thrombin play a key role locally in the cessation of menstrual bleeding through instigation of the coagulation factors. On the other hand, fibrinolysis prevents clot organisation within the uterine cavity while plasminogen activator inhibitors (PAI) and thrombin-activatable fibrinolysis inhibitors control plasminogen activators and plasmin activity. Abnormalities of uterine bleeding can result from imbalance of the haemostatic factors.
Endometrial haemostasis is a vital process for the control and cessation of menstruation. It is influenced by systemic clotting systems that react to the hormone induced shedding of the endometrium, forming the haemostatic platelet plug and fibrin deposition. Local factors within the endometrium are paramount to ensure the process is highly regulated, with adequate blood vessel occlusion, breakdown of thrombus, and tissue regeneration.
Davies, J., & Kadir, R. A. (2012). Endometrial haemostasis and menstruation. Reviews in Endocrine and Metabolic Disorders, 13(4), 289-299. doi:10.1007/s11154-012-9226-4

Coagulation & Fibrinolytic Pathway in 5 minutes - Simple and Easy

Fibrinolysis Wikipedia


Is there any virus that contains both DNA and RNA in its genome? It is known that viruses contain DNA or RNA- either one and not both.

I came across a question: Which virus contains both DNA and RNA?

Here is the results summary of the study that describes the discovery of DNA:RNA hybrid virus:

  Results
  Bioinformatic analysis of viral metagenomic sequences derived from a hot, acidic lake revealed a circular, putatively single-stranded DNA virus encoding a major capsid protein similar to those found only in single-stranded RNA viruses. The presence and circular configuration of the complete virus genome was confirmed by inverse PCR amplification from native DNA extracted from lake sediment. The virus genome appears to be the result of a RNA-DNA recombination event between two ostensibly unrelated virus groups. Environmental sequence databases were examined for homologous genes arranged in similar configurations and three similar putative virus genomes from marine environments were identified. This result indicates the existence of a widespread but previously undetected group of viruses.

And here is the link to the paper:

https://biologydirect.biomedcentral.com/articles/10.1186/1745-6150-7-13


Can foraging worker ants become queens? The TL;DR is in the subject line.

The back story is as follows:

I am combating ants in my high-rise apartment unit.  One piece of online advice was to vacuum regularly, and pay attention to edges of the floor.  I'm considering taping up the vacuum bag opening so that I'm not tossing out a nearly empty bag every day (almost $10 each, and terrible for the environment).

I had initial concern that an ant might get out, so I also double bagged the vacuum bag.  This is making it inconvenient to use, meaning that I'll vacuum less.  Exactly the opposite of the online advice.

I think that the chances of an ant getting out is low, and even if escape was possible, it probably wouldn't be more than one or two.  I can accept that, if they don't go start generating more ants.

I haven't found anything online about workers being able to transform into queens once they are foraging.  What I have read is that it has to do with genetics and extra nutrition during the formative stages.

Is it safe to assume that foraging workers will not be able to turn into queens?

After note: It just occurred to me, if ants can chew through wood, then there's nothing to prevent them from chewing through a vacuum bag over the course of a day or more.  They can even chew through the plastic bag in which the vacuum bag is sealed.  Is this a valid concern, or is their ability to chew limited to wood?

Workers cannot become "queens" because the development of a larvae into a worker or a queen happens during the larval stage, depending mainly on the food that the larvae is given. That being said, once the queen is removed from a colony, in many ant and other social hymenopteran species, workers are no longer inhibited by the pheromones produced by the queens that prevent the growth of the worker's ovaries. So in some species, removing the queen can lead to workers laying eggs but these will most often be parthenogenetic and lead to the production of males only. In a very few species, workers can produce female eggs by parthenogenesis or mate with males after the removal of the queen. Such traits are usually associated with large body size and small colony size, so they are unlikely to be the ones you have in buildings.

  Bourke, A. F. (1988). Worker reproduction in the higher eusocial
  Hymenoptera. The Quarterly Review of Biology, 63(3), 291-311.

  Page, R. E., & Erickson, E. H. (1988). Reproduction by worker honey
  bees (Apis mellifera L.). Behavioral Ecology and Sociobiology, 23(2),
  117-126.

  Wenseleers, T., Helanterä, H., Hart, A., & Ratnieks, F. L. (2004).
  Worker reproduction and policing in insect societies: an ESS analysis.
  Journal of evolutionary biology, 17(5), 1035-1047.


How to find the concentration of an enzyme? I need to know the concentration of pectinase enzyme (sigma aldrich) which has stated 5KU, 5U/mg protein (lowry) and lot result 20U/mg protein in the label of the enzyme bottle. This is all it has mentioned. How do I calculate the concentration from this information?

hey everyone, sorry for not been straight forward with the question. I'm asking for U/ml.I think I can find it if the label has mentioned the total volume of enzyme solution. Then I know how much mg in 5KU which is 5000/5=1000mg which I can easily get mg/ml or U/ml which is more appropriate. But the question is pointless if it hasn't mentioned the total volume. Thanks for the replies.

PS My supervisor asked me to prepare a standard graph of enzyme concentration (U/ml) against absorbance(O.D value) for DNS assay instead of a Galacturonic acid standard/glucose standard.(galacturonic acid is not available so he thinks it is better to you this graph than a standard glucose graph)

PS I checked the bottle again.On the bottom side of the bottle it has stated 10 ml.

The enzyme unit U is not a measure of enzyme concentration but its activity, defined as the conversion of 1 micro mole ($\mu$mol) of substrate per minute (under a specified condition such as pH and T)

Absorbance (O.D. at a specific wavelength) of the enzyme is a measure of enzyme concentration, regardless of its activity. Depending on the unit of the extinction coefficient, Absorbance can be converted directly by Beer's Law to enzyme concentration, typically in mg/mL or in the standard mM.

5U/mg is the specific activity of pectinase, and the bottle contains a total of 5 kilo U (KU), meaning that there is a equivalent of 1g of active enzyme inside (often in powder). If you assume all pectinase molecules inside the bottle are active, when dissolved in 10mL water, you have a pectinase concentration of 100mg/mL. If the molecular weight of pectinase is 31kDa (31000 g/mol), then 100mg/mL is the same as 3.2mM in concentration.

By the way, Lowry protein assay measures enzyme concentration, not activity in U while DNS assay measures activity (of enzyme that produces reducing sugars), not enzyme concentration.


Is there a strong reason to be sceptical about the "cured HIV patient" being reported by mainstream media? There's a story going round the news about a baby that was, apparently, cured of HIV using a cocktail of drugs at an early age. The story piqued my interest, but details seem scarce. One of the main things I've seen being refuted about it is the difficulty in proving that (a) the child actually had HIV in the first place, and (b) that the child is really fully cured.

I've seen the following arguments:

Western Blot tests on young children are practically useless, since they test for antibodies. The child will likely have antibodies passed down by the HIV+ mother, regardless of whether the child has HIV. The test will show the antibodies, which may be mistaken for an active immune response from the child. As such, there will be a high false-positive rate on such tests.
Using PCR to attempt to identify a lack of HIV is flawed, since it may lay dormant in a small colony of T-cells until a secondary immune response is triggered, causing a new outbreak. As such, it is very difficult to show that a patient is "cured".

Are these arguments valid? Are there any other reasons to be sceptical about the claims? Any other information that might be useful in understanding the case?

Western Blot tests on young children are practically useless, since they test for antibodies. The child will likely have antibodies passed down by the HIV+ mother, regardless of whether the child has HIV. The test will show the antibodies, which may be mistaken for an active immune response from the child. As such, there will be a high false-positive rate on such tests.

The mother's T-cells are in the fetus anyways (15/18 Fetal lymph nodes contained T-reg cells). If the mother is infected and left untreated (as was the case with the Mississippi child), then the viral load is going to be high. The chances of it passing to the fetus are going to be high as a result.

  Using PCR to attempt to identify a lack of HIV is flawed, since it may lay dormant in a small colony of T-cells until a secondary immune response is triggered, causing a new outbreak. As such, it is very difficult to show that a patient is "cured".

"Flawed" is the wrong word. The argument is built into the tests. The child was functionally cured - which means below detectable levels instead of "completely nonexistent within the body." Yes, it's entirely possible that the HIV is hiding somewhere in the body and could make a reappearance, but at the moment it's not showing any significant titer in the blood.

I don't think there's been a test developed that can assess the entire body to find the lone viral capsid left.

However - that isn't to say that the "cure" has been found. So far it's a single person cured this way. One. That makes it a case study, not a therapy, treatment, vaccine, or even reasonable assessment that it's possible to do again.


Why don't teeth glow? Hydroxyapatite is the main component of tooth enamel. It contains phosphorus in the form of phosphates, pyrophosphates etc. that are found to exhibit the the property of phosphorescence. But why don't the teeth glow in the dark ?

Just to add a little more on the interface between optics and dentistry:

Whilst teeth do not phosphoresce, they do in fact autofluoresce.

The differential auto fluorescence of healthy tooth and carious tooth has been used for the early diagnosis of caries. (Gugnani N, Pandit IK, Srivastava N, Gupta M, Gugnani S. Light induced fluorescence evaluation: A novel concept for caries diagnosis and excavation. J Conserv Dent 2011;14:418-22 and http://www.opticsinfobase.org/boe/abstract.cfm?uri=boe-2-1-149)

To see teeth glow, or rather, fluoresce, they should be illuminated with short wavelength light, like blue light (wavelength 450 nm) and the teeth will glow green, which will be visible if the the blue light is filtered out.

Also see http://www.inspektor.nl/dental/qlfmain.htm#QLF%99%20Basic%20Principle


How much solar energy required to produce enough food for a herbivore? Just curious, how much solar energy can power an (herbivore) animal? Specifically, is it enough sunlight on Mercury (4 to 10 times brighter than on Earth) to "feed" a zebra? Will it be sufficient at 10x more effective photosynthesis? Notwithstanding lack of atmosphere, water being buried deep and other minor problems, of course :)

BMR is the amount of energy an animal uses just by being alive. BMR is proportional to the mammal's mass. More precisely, BMR is proportional to the mammal's mass to the power of 3/4. A 0.1 kg mammal has a BRM of 10 kcal/day. Using the aforementioned proportionality, a 600 kg mammal (average mass of a cow) will have a BMR of 6800 kcal/day.

This means that a cow would need about 6800 kcal of energy per 24 hours (1440 minutes) to survive.

The amount of solar energy reaching the earth’s atmosphere is 0.00194 kcal per square centimeter per minute. Since, according to you, there is 10 times the amount of sun in Mercury, there should be 0.0194 kcal per square centimeter per minute hitting Mercury.

0.0194 kcal per minute for 720 minutes (counting only day - since there is no sun during the night) equals 13.968 kcal per square centimeter per day. At this rate, the sun has to hit an area of 486 cm squared to harness enough energy for the cow.

In summary:

The average cow needs about 6800 kcal per day to survive (BMR)
On Mercury, the sun generates 13.968 kcal per square cm
Which means, the sun has to hit an area of 486 square cm

In one sentence:

In order to power a cow on Mercury, the sun has to hit an area of 486 cm for 720 minutes.

To learn more:

https://en.wikipedia.org/wiki/Kleiber%27s_law
http://www.ase.org/resources/measuring-number-calories-sunlight


Is global life expectancy normally distributed? I am trying to find out what the world distribution of life expectancy looks like.

Let's see!

I took the most recent WHO data from here and did a quick an dirty analysis in R. Here is the histogram as well as a normal distribution with the same mean and standard deviation as the actual data:

Does not look very normally distributed. In fact, the shapiro test confirms this impression:

Shapiro-Wilk normality test

data:  df$life_expectancy
W = 0.94141, p-value = 4.338e-07

Edit

Commenters requested to weigh the life expectancy by population size.Well...the result is pretty ugly:

Apparently, the large populations of China and India produce disproportionally high bars. However, keep in mind that we are using averaged values which means that all the variation within countries is not represented by the histogram. We would actually have to have data on individual ages of death for a random sample of the world population to finally settle the question. :/


With an excess supply of nutrients, in how much time will the number of algae cells double in full sun? I am growing algae in a container in my roof. It gets sunlight for the whole day as there are no clouds nowadays in my area. There is no lack of nutrients as I am constantly adding water mixed with organic waste. The dirt visible in the photos is from manure, which I added as fertilizer.

The water has become green and algae is deposited on top covering almost all of the surface area. There are no threads, so it is not filamentous.

Since there are probably several species of algae in it which I don't know, please tell me the doubling time of most common species.

Also, is there anything I can do to hasten the production of cells?

Below are pictures of the culture, as asked for.

I am presuming this a Blue Green Algae(BGA) Identify your algae

Growth rate for N1, N2 biomass at t1 and t2 time respectively, can be give as,

Growth rate:   $K'$ = $\ln \frac{(N_2 / N_1)}{(t_2 - t_1)}$

(source: csiro.au)

So, by this equation it is easy to calculate the doubling time of your algae.

For BGA normal doubling time is 6-8 hours.$^2$

Source:

http://www.marine.csiro.au/microalgae/methods/Growth%20rate.htm
https://sites.google.com/site/biomonstaaar/algae


How efficient is the sodium-potassium pump ? I am reading about transportation of ions in a cell. It is necessary to transport sodium back out and potassium back in, against their electrochemical gradient. This task is carried out by sodium-potassium pump, which transports three sodium ions out across the cell membrane for every two potassium ions carried inward. The source of energy for this transport process was hydrolysis of ATP.
My question is the order of the efficiency of a sodium-potassium pump.

I couldn’t find a value for this but I have calculated an efficiency of 84 %.

Caution: this seems too high to me, so I show below my calculation, fully dissected, in case anyone can spot an error.

First of all the parameters. Ion concentrations are:

internal Na+  = 12 mM;
external Na+ = 140 mM;
internal K+   = 140 mM;
external K+   = 5 mM;
and membrane potential = -60 mV (negative inside the cell)

I use these values for constants:

gas constant R = 2 cal mol-1 K-1

Faraday constant F= 23,062 cal mol-1 V-1

and I’ll assume that the temperature, T, is 37℃ = 310 K

The Na+/K+ pump uses 1 molecule of ATP to move 3 Na+ out of the cell and 2 K+ into the cell.

The efficiency of the pump is therefore energy required to move these ions/energy released by ATP hydrolysis.

How much energy is needed to move the ions? In each case there is a contribution from the fact that the ions are moved along a concentration gradient (the chemical component) and from the movement along a gradient of electrical potential (because of the membrane potential). In considering these components we have to think about whether energy needs to be used when the ions move, or whether it is released.

For the chemical component we can use

ΔG = RTln(ratio of concentration, inside and outside), where ln = natural logarithm

The sign of this ΔG will depend upon how the ratio is expressed (in/out or out/in) but the absolute value will be the same. So I calculate an absolute value and then deduce the sign of the free energy change.

For Na+ ΔG = modulus(2 x 310 x ln(12/140)) = 1,523 cal mol-1

Since Na+ is being moved up a concentration gradient, work must be done so the free energy change is positive.

Applying the same formula and logic for the movement of K+:

ΔG = 2,065 cal mol-1.

(This value is greater than that for Na+ because the concentration gradient is steeper.)

Next I calculate the electrical components using:

ΔG = zFμ, where z is the charge on the ion = 1 in both cases, and μ is the membrane potential (in volts).

So for both Na+ and K+ the absolute ΔG value is 1,383 cal mol-1.

However, in this case Na+ is moving out, against the membrane potential while K+  is moving in with the membrane potential. Thus the value of ΔG is positive for Na+ but negative for K+.

Now I combine the chemical and electrical components to get the net free energy change per mole of ions moved:

Na+ ΔG = 1,523 + 1,383 = 2,906 cal mol-1

K+ ΔG = 2,065 -1,383 = 682 cal mol-1

Now I consider how to combine these two values. Each cycle of the pump uses 1 ATP to move 3 Na+ and 2 K+. Therefore each mole of ATP moves 3 moles of Na+ and 2 moles of K+.

Energy from 1 mole ATP that is used to move ions = (3 x 2,906) + (2 x 682)

= 10,082 cal

= 10.1 kcal

How much energy is released from hydrolysis of 1 mol of ATP? Estimates vary but a commonly accepted value seems to be 12 kcal mol-1

Thus efficiency of the pump = 100*(10.1/12) = 84%.

I think the free energy of ATP hydrolysis depends on ATP concentration, pH, and the amount of Mg present. For a well-fed brain, this is around 14 kcal/mol (see Journal Of Physical Chemistry B, 114 (49), (2010). 16137-16146). This gives an efficiency of around 70%, still too high but better...


Identification of colorful jelly-like marine creature I'm on my trip to Gulangyu Island, Xiamen, Fujian Province, China. Today(4th February 2017), at 12:13 A.M., I saw a beach filled with huge rocks on which various dry shells reside. Among the shells, I spotted a colorful eye-like marine creature.

To figure out what it is, I torn it off from the rock. (don't worry, I've put it back after taking this photo)

Also, it feels soft when I poked it, and its size is approximately 1cm to 1cm. Could anybody please tell me what I've found?

You've found a sea anemone, a cnidarian of the Order Actiniaria. In this case, the anemone is closed and thus hiding its characteristic tentacles (likely as a form of protection while "out to dry").

I'm by no means an expert, but my guess is that you have a vertically striped species of the genus Diadumene  -- likely one of many morphotypes of the species Diadumene lineata. I've seen common names vary between "Striped Sea Anemone", "Lined Bead Anemone" and "Orange-Striped Green Anemone."

Here is a double stripe specimen from San Francisco, and below are two pictures from Ria Tan's Wild Singapore image collection:

Closed:

Open:

Without further information regarding size, color/length of tentacles etc., it will be hard for a non-expert like me to find a better answer.

According to the Wikipedia page, this species originated along the Pacific coast of Asia, but has spread more globally from attachment to ship bottoms, oyster shipments, and seaweed farming.

It appears from your picture, that you found your specimen out of water along the shoreline. Again according to the Wikipedia  article:

  Diadumene anemones display high tolerance to inter-tidal exposure and drying out in extreme summer heat


Why is aconitase classified as a lyase? Aconitase in the TCA (tricarboxylic acid) cycle isomerizes citric acid to isocitric acid via cis-aconitic acid intermediate. Since overall it functions as an isomerase, why it does not belong to isomerases? Why does it belongs to lyases even though it involves hydrolysis of water in second step? Should not it belong to hydrolases in this way?

So how it can be justified that it is lyase?

The mechanism of aconitase classifies it as a lyase, even if (under most physiological conditions), the relative concentrations of substrates results in it catalysing the conversion of citrate to isocitrate. In short, it is not an isomerase because the substrate is released after each step.

As the mechanism of aconitase shows, it catalyses the conversion of either citrate or isocitrate to aconitate as well as the reverse reaction. This is similar to the reaction catalysed by fumarase, which is the reversible addition or removal of water.

Because the physiological concentrations of citrate is much larger than the concentration of isocitrate, aconitate overall catalyses the reaction from citrate to isocitrate, and does so via catalysing two lyase reactions.

Regarding the other part of your question, hydrolases are a completely different group of enzymes, and none of the reactions involved are related to hydrolases in any way.

While water is in fact involved in the reaction, it is not used in the process of hydrolysis. To be specific, hydrolysis (in its enzymatic/organic, and not chemical sense) requires that water be a substrate required to break apart a molecule A-B into A and B. Since the addition of water does not result in citrate/isocitrate being broken down into two smaller organic molecules, aconitase is not a hydrolase.


What is PDMS used for in microfluid models? When creating a microfluidic model using polydimethylsiloxane (PDMS), is PDMS used only as a stamp to lay proteins in coverslips or can cells be cultured in the channels or patterns created in PDMS?

PDMS in fact is established to be a reliable material for cell culture in many microfluidic devices.
Here are several papers (Titles) including reviews on microfluidic devices using PDMS:

Microfluidic devices for cell cultivation and proliferation Here
Adhesion patterns in the microvasculature are dependent on bifurcation angle Here (Microvascular Research)
Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices Here
A physiological model of the tumor microenvironment for screening drug delivery systems Here (Cancer Research Proceedings)
A Novel Dynamic Neonatal Blood-Brain Barrier on a Chip Here (Plos One)
A novel microfluidic assay reveals a key role for protein kinase C δ in regulating human neutrophil–endothelium interaction Here(JLB - Journal of Leukocyte Biology)

My research at Temple University is actually based on a microfluidic device to study cell-cell interaction in a real-time fashion.
Majority of established protocols are using fibronectin or collagen, and in the case of tumor cells culture, matrigel for extracellular matrix. Devices are being coated with the ECM protein and then cells can be cultured in the device.

We have shown that in our microfluidic device, human umbilical vein endothelial cells (a.k.a. HUVECs) are forming a complete 3D lumen in the channels.

It is important to note that due to the elasticity of the PDMS, cells are needed to be under physiological flow conditions. Otherwise, it was shown that the cells start to detach from the PDMS. Under such physiological conditions (flow, media, gas exchange), cells can be alive and functional up to a week or two, depending on the design. In our system, the last two publications, cells are functional up to 2 weeks and 1 week respectively.


What allows for no selection pressure against non-coding DNA? Genomes of prokaryotes tend to have a higher proportion of coding DNA to non-coding DNA in their sequence than eukaryotes.
This means that either eukaryotes accumulated lots of non-coding DNA, much of which is regulatory, in the course of evolution — or that prokaryotes lost this proportion of non-coding DNA.

In order for transcription of a particular gene to occur, it seems that there must be some search performed on the DNA sequence to find the gene to be transcribed.
This search seems to be independent of the sequence length — if it weren't, that could act as a selection pressure, inducing loss of DNA that doesn't do anything. Therefore, it seems that the method by which this search is done must be O(1) — that is, it takes a constant amount of time to complete the search, regardless of the length of the sequence.
Such a method would be most welcome in computational sequence searches (in place of BLAST for example).

My question is, what, if anything, is currently known about the process of transcription that could explain this constant search time?
Or, if I am completely wrong in my assumptions, what are more reasonable assumptions?

A) Here's the thing about evolution - just because something might have an adaptive benefit does not mean that trait will evolve ; this is contingent on the occurrence of the necessary mutations to produce a phenotype in the first place.

B) There are multiple copies of the transcriptional machinery (RNA pol, Transcription Factors) - numbering in the tens of thousands when you are talking of the core transcriptional machinery.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC84381/

The 3d structure of chromatin , as well as the large amounts of the core transcriptional machinery, prevent there from being too much adaptive benefit to losing large amounts of noncoding DNA.

C) Thirdly, there are suggestions that enhancer promoter contacts, which can greatly facilitate transcription, may be in some part at least determined by sequence motifs in the loops that bring together enhancers and promoters ; so there would be costs associated with losing ncDNA en masse. See www.nature.com/ng/journal/v48/n5/abs/ng.3539.html

D) Indeed, even when one looks at repeat elements derived from endogenous retroviruses, there are those that are of functional significance; Syncitin (which actually codes for a protein) is responsible for trophoblast fusion + placenta formation (See https://www.ncbi.nlm.nih.gov/pubmed/10693809 )  , and there are multiple repeats that themselves serve as enhancers of interferon response genes. See https://www.ncbi.nlm.nih.gov/pubmed/26941318


Age of shells on beach As a child I watched tiny fiddler crabs living in conical shells, and many years later I find that people study fossil turritellids. So now I wonder: how old are shells, typically, that you see on beaches? Do they wear out after a year of sand abrasion? 100 years, a million? Is it known?

Mollusk shells found on typical east coast (US) beaches can range from days old (the animal that made the shell died recently) to thousands of years old.  Some shells in our state, North Carolina, have been dated as 40,000 years old.  A high number of "seashells" found on east coast beaches are from mollusks that lived in the marsh on the back side of the island.   The presence of these shells on ocean beaches provides evidence of island migration - the island has moved landward over the marsh until what was once the marsh is now the ocean shore.  Once buried in the sand, the shell is well preserved until erosion uncovers it.  Occasionally, fossilized shells are washed up on beaches after having been dislodged from offshore limestone deposits - these shells can be millions of years old.

-Richard -
Carolina Ocean Studies


Why can't we kill ourselves by holding our breath? Is it possible to kill yourself by holding your breath?

This question is obviously copied from Quora, but I had heard it as a fact that we cannot kill ourselves by holding our breath and I'm looking for a referenced answer.

Short answer
Healthy people cannot hold their breaths until unconsciousness sets in, let alone commit suicide.

Background
According to Parkes (2005), a normal person cannot even hold their breath to unconsciousness, let alone death. Parkes says:

  Breath‐holding is a voluntary act, but normal subjects appear unable
  to breath‐hold to unconsciousness. A powerful involuntary mechanism
  normally overrides voluntary breath‐holding and causes the breath that
  defines the breakpoint.

Parkes explains that voluntary breath‐holding does not stop the central respiratory rhythm. Instead, breath holding merely suppresses its expression by voluntarily holding the chest at a certain volume. At the time of writing, no simple explanation for the break point existed. It is known to be caused by partial pressures of blood gases activating the carotid arterial chemoreceptors. They are peripheral sensory neurons that detect changes in chemical concentrations, including low oxygen (hypoxia) and high carbon dioxide (hypercapnia). Both hypoxia and hypercapnia are signs of breath holding and both are detected by the chemoreceptors. These receptors send nerve signals to the vasomotor center of the medulla which eventually overrides the conscious breath holding.

The breaking point can be postponed by large lung inflations, hyperoxia and hypocapnia, and it is shortened by increased metabolic rates.

Reference
- Parkes, Exp Physiol (2006); 91(1): 1-15


How can they know that they found "all the PERVs"? (porcine endogenous retroviruses) The New York Times article Gene Editing Spurs Hope for Transplanting Pig Organs Into Human links to the recent Science publication Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9
 Niu et al. Science  10 Aug 2017: eaan4187, DOI: 10.1126/science.aan4187.

The highlighted section of the abstract says "...we inactivated all the PERVs in a porcine primary cell line..." where a PERV is a porcine endogenous retrovirus, an RNA virus which carries its own reverse transcriptase molecule and converts to DNA before incorporating into the host's genome.

Question: Do they mean to say all currently known PERVs or is there some way to be absolutely sure that every PERV in the pig's genome is known. In other words, could they have missed one? Could there be an as-yet unidentified PERV? I don't really want to ask about the possibility of an entirely new and unknown class of retroviruses; rather I'm trying to understand how all PERVs as currently defined can be identified in a pig cell line.

The reason that this is important is that the motivation here is to generate pig organs for transplantation into humans. The concern is that the pig genome may contain retroviruses that could then infect the human host, and the further possibility that it could be passed infectiously to others. As pig to human infectious disease is always a concern, starting with a "clean" pig genome is of particular epidemiological importance.

The first sentence of the NYTimes article:

  In a striking advance that helps open the door to organ transplants from animals, researchers have created gene-edited piglets cleansed of viruses that might cause disease in humans. (emphasis added)

Abstract of Niu et al. 2017:

  Xenotransplantation is a promising strategy to alleviate the shortage of organs for human transplantation. In addition to the concern on pig-to-human immunological compatibility, the risk of cross-species transmission of porcine endogenous retroviruses (PERVs) has impeded the clinical application of this approach. Earlier, we demonstrated the feasibility of inactivating PERV activity in an immortalized pig cell line. Here, we confirmed that PERVs infect human cells, and observed the horizontal transfer of PERVs among human cells. Using CRISPR-Cas9, we inactivated all the PERVs in a porcine primary cell line and generated PERV-inactivated pigs via somatic cell nuclear transfer. Our study highlighted the value of PERV inactivation to prevent cross-species viral transmission and demonstrated the successful production of PERV-inactivated animals to address the safety concern in clinical xenotransplantation. (emphasis added)

You are right, they should have written "all currently known PERVs".
Although the structure of ERVs is generally well understood (https://www.ncbi.nlm.nih.gov/pubmed/26104559; https://www.ncbi.nlm.nih.gov/pubmed/26818261) there may be some complex cases that have been missed. Also, the map of the porcine genome is not fully complete yet (nor the human one) so you cannot be sure that there are no ERV-derived sequences hidden in the unsequenced regions.


Western blot transfer issues I am having some issues with transfer efficiency to nitrocellulose membranes. To get all the technical info out of the way:

Transfer method: Wet, tank transfer overnight at 30V with normal tris-glycine transfer buffer + 20% methanol. I do this at RT with stirring. Of note: I have not recently been equilibrating my gel in transfer buffer, as this was reported to decrease transfer of basic proteins due to the stripping of SDS (I can furnish this reference if anyone is interested).

Sample: In all cases these are in vitro reconstituted nucleosomes with or without certain enzymes--the main transfer issues are the core histones, which are highly basic (pI 10-11). There should be no more than about 100 ng of each histone being transferred per lane. The salt in the sample is <25 mM. They are boiled prior to loading. There is no noticeable precipitate in the samples before or after boiling, however the volume is quite small (20 ul) so I would not necessarily detect precipitation.

Nitrocellulose is 0.2 um and is pre-equilibrated in transfer buffer prior to transfer.

The gel: I use a 15% gel, 1.0-1.5 mm thick. The acrylamide I use is 37.5:1 acrylamide:bis. Some issues could arise here--the acrylamide solution is about 1 year old and has been stored at RT that entire time (which is what is recommended on the label). In addition, we typically keep a stock of 10% APS at 4C, so I cannot guarantee my APS has not gone bad.

I have been quite careful generally to ensure there are no bubbles between any components of the sandwich and filter paper/foam pads are soaked in transfer buffer.

Now to the issue. Attached are images of the membrane after transfer (and also after processing, thus the relatively high background from the blocking buffer) as well as the gel after transfer. As you can see from lane to lane I seem to have differing transfer efficiencies. This has occurred with my last three blots. My first thought was sample precipitation, but I have not had this issue before despite running essentially the same blot many many times over the past year.

Any thoughts? Quite frustrating, as it has appeared suddenly with identical protocols/reagents as before.

The first thing that comes to mind for me is that the transfer seems excessive... isn't histone sub-20 kDa? I have often transferred similar-sized proteins at 4C for 30 min, 30V.

In general, presence of SDS will inhibit transfer efficiency, and increasing alcohol in your TG buffer will counteract the effect of SDS present. Perhaps bring down the methanol to 10%? I would be curious to see that reference regarding the SDS/pre-equilibration, though. That certainly runs contrary to how I understand it working. (Note: I flip-flop between equilibrating or not equilibrated my gels without any observable differences.)

If your acrylamide or ACS had gone bad, your gel wouldn't set.

If you don't already, I'd suggest running a Bradford or BCA on your samples to be sure that you're loading the same amount into each well. Having loaded more into each well could give the appearance of uneven transfer.

To be honest, I don't really see what you mean by uneven transfer, based off the Coomassie and Ponceau. If you're seeing a gradient effect across a large portion of the gel, I would suggest you visually inspect to make sure that the gels you cast are even width all the way across. Having a thicker gel in one place than in another could impact the resistance (heat) in that part of the gel and impact transfer. If you're seeing a lane-by-lane transfer efficiency shift, I would suggest localized pH or temperature changes due to uneven loading of salts, protein concentration, or lipid concentration.

Hope this helps!


What is a topological domain? I found this definition on Wikipedia, but I didn't quit undestand it :

  Topologically associating domains (TADs) are genomic regions ("chromosome neighborhoods") used to summarize the three-dimensional nuclear organization of mammalian genomes.

Can anyone explain it more, and give examples if it's possible ?

TADs were initially discovered by computing contact probabilities between regions of the genome using HiC (a chromosome conformation capture method, that try to provide an idea on how the genome is organized inside the nucleus by computing the probability of each contact to be located nearby another locus). People have found that instead of being random, some loci were in contact with some regions of the genome rather than the others, implying some form of functional significance.

In short: a TAD is a genomic region of increased contact probability. They are of functional relevance (for instance, Enhance-Promoter gene expression regulation occurs primarily within one TAD rather than between two (adjacent TADs).

You might be interested in the following papers:

A 3D Map of the Human Genome at Kilobase Resolution Reveals Principles of Chromatin Looping (might be behind paywall)
Topological Domains in Mammalian Genomes Identified by Analysis of Chromatin Interactions

Finally, the following picture (extracted from the latter publication) provides an example of two TADS:

The bottom panel shows the contact probability: two regions of the genome appear to be isolated whereas they are nearby. They show significant self-interaction. One interpretation for that (top panel) is to imagine that the genome exhibit some reproducible condensation, explaining the increased contacts.


Why do animal cells "mistake" rubidium ions for potassium ions? So, I was browsing the Wikipedia article for rubidium, and came across this interesting tidbit:

Rubidium is not a known nutrient for any living organisms. However, rubidium ions have the same charge as potassium ions and are actively taken up and treated by animal cells in similar ways.

This immediately struck me as interesting, and a completely wrong explanation, as all singly-ionized ions have the same net charge.
Now, from my basic understanding of chemistry, elements of the same group have the same valence electron structure, which accounts for much of the element's chemical effects. And if you look at the periodic table, sure enough, rubidium falls below potassium, in the Group 1 alkali metals.

But, from my old memories of basic biology, even saying "these elements are vertically adjacent on the periodic table" is an incomplete explanation, because, for example, the body has no problem "telling apart" sodium from potassium, or plenty of other vertically adjacent elements.
A survey of other potentially relevant features reveals the calculated radius of rubidium (265 pm) is relatively close to the radius of potassium (243 pm), and the first ionization energy of rubidium (403.0 kJ/mol) is also pretty close to that of potassium (418.8 kJ/mol). So they do seem fairly similar, but question still remains, how "similar" does an element need to be to become a biological proxy?
This leads to me to the following questions:

Why does rubidium act as a biological proxy for potassium? Also:

Why is cesium also a decent, but less good, biological proxy for potassium?
Why is potassium not a biological proxy for sodium?

If rubidium acts as a biological proxy, why is it still not a "nutrient" and not considered to play a biological "role"?
Which other sets of elements can act as biological proxies for each other? (I presume expanding this question to molecules yields too many answers to be useful.)
Is this rubidium-potassium proxy effect specific to animals or to all life?

Some good references I've found, skimming the literature (I started by looking at some references in this article):

Relman AS. The physiological behavior of rubidium and cesium in relation to that of potassium. Yale J Biol Med. 1956;29(3):248–62.

Relman notes that this phenomenon was first noted by Sydney Ringer in experiments begun 1882, published in the Journal of Physiology in the year 1884 (or so).

Fieve RR, Meltzer H, Dunner DL, Levitt M, Mendlewicz J, Thomas A. Rubidium: biochemical, behavioral, and metabolic studies in humans. Am J Psychiatry. 1973;130(1):55–61.

But I'd rather someone present an intuitive explanation for this before or after I attempt to dive in.

There are multiple biological mechanisms that can be brought to bear for distinguishing between atoms. In addition to binding properties (e.g., ionic charge, electronegativity, bond strength), there is also the size of the atoms and even their vibrational properties.
For one of these mechanisms to actually be used, however, it needs to be evolutionarily selected for in some way. The binding properties are by far the simplest to select for, since they are fairly local and permissive in nature, while the others require much more carefully tuned mechanisms.
Thus, we should expect to see most biological selectivity to be developed in terms of binding properties, as seems to be the case. For same-group covalent-binding such as one encounters with oxygen vs. sulfur, there are major differences in the properties of the bonds, giving ample opportunity for biological selectivity.
For Group 1 alkali metals like sodium, potassium, and rubidium, however, ionic bonding is much more uniform. Thus, we should expect mechanisms that apply to one to apply to all unless strongly selected for distinguishing.  In the case of potassium versus sodium, which are both very important in biochemistry, a recently discovered mechanism in sodium-potassium pumps uses binding properties to grab both, then atomic size (via steric hindrance) to distinguish sodium versus potassium as described nicely in the answers to this question.
Presumably, there simply has not been sufficient evolutionary pressure to establish mechanisms for distinguishing rubidium from potassium in the same way.  While sodium and potassium have approximately equal abundance, rubidium is approximately two orders of magnitude less abundant than either. Cesium, in turn, is nearly two orders of magnitude less than rubidium, and rapidly decaying francium mostly doesn't exist.
Thus, unless there is a particularly problematic effect of occasional incorporation of rubidium or cesium, then, we should expect these elements to be essentially indistinguishable from potassium biologically, but not from sodium, since sodium/potassium distinguishing mechanisms will reject them all along with potassium into the "not the right size to be sodium" bin.


Why does pETDuet-1 only have one T7 terminator but two T7 promoters? Why does pETDuet-1 only have one T7 terminator but two T7 promoters (vector map below)? Is there any advantage of having only one terminator? There are going to be two mRNAs transcribed from this vector: one polycistronic (with two genes) and one monocystronic. Also, presumably, there will be more RNA for the gene expressed from the second promoter compared to the gene expressed from the first. That could lead to higher expression of the second protein, provided that the RBSs are the same. Is there experimental evidence confirming/disproving that hypothesis?

Is there any advantage over using polycistronic mRNA vs moncystronic? Have Duet vectors been used for heterologous expression of long (>40kb) biosynthetic pathways?

I think the presence of a single terminator is related to the high processivity of T7 RNA polymerase.

T7 RNA polymerase will transcribe a circular plasmid repeatedly before dissociating. The T7 terminator after ORF2 ensures that the transcript terminates efficiently.

Based on your schematic, these are the potential mRNA transcripts from that construct:

Transcript from T7 promoter #1

 5'---RBS---ORF1---STOP----RBS---ORF2---STOP---[T7 terminator]---3'

Transcript from T7 promoter #2

 5'---RBS---ORF2---STOP---[T7 terminator]---3'

It is doubtful that this vector will produce precisely equimolar concentrations of each protein product, however, the topology ensures that there is at least some of each protein produced. Of course, this could be tested empirically.

I hope that helps!


Why does renal artery stenosis lead to activation of Renin-Angiotensin system? To my current understanding, stenosis or narrowing of the arteries would mean higher blood pressure, similar to atherosclerosis. So the Renin system should be suppressed to counter the high blood pressure. But..

I have read in a textbook that renal artery stenosis means lower blood flow in the kidney due to the narrowing of the artery. Thus the Renin system is activated to increase blood pressure.

So why does narrowing of the renal artery cause a decrease in blood pressure, and not increase?

So why does narrowing of the renal artery cause a decrease in blood pressure, and not increase?

It doesn't. It causes an increase.

If the blood flow to the kidney is decreased for any reason (for example, hemorrhage or dehydration), the kidney is one of the organs that will protect it's perfusion by causing an increase in BP through the renin-angiotensin-aldosterone system (RAAS).

Very briefly: decr. blood flow -> renin secretion by juxtaglomerular cells -> conversion of serum Angiotensinogen into Angiotensin I (+ ACE) -> Angiotensin II => elevated BP (by a host of mechanisms).

If there is renal artery stenosis, the kidney will try to compensate for this (locally) reduced blood flow by activating the RAAS. The kidney doesn't 'know' that the blood flow/pressure is only locally reduced. It will cause systemic hypertension as a way to increase it's blood flow.

Atherosclerosis is a whole different ball of wax.


Are penguins plantigrade or digigrade? I'm trying to rig a 3D model of a penguin, but I don't know where to put the bones near the ankle because I can't tell if they're digigrade or plantigrade. Nearly all birds are digigrade, but penguins spend a lot of time walking and don't generally grasp or run with their talons.

Plantigrade.

  The penguins are highly specialized for their flightless aquatic
  existence. The feet are located much farther back than those of other
  birds, with the result that the bird carries itself mostly upright;
  its walk can thus be described as plantigrade (i.e., on the soles).
  The sole comprises the whole foot instead of just the toes, as in
  other birds. The most notable characteristic of the group is the
  transformation of the forelimb into a paddle. This is accompanied by a
  body morphology particularly adapted to movement in a liquid medium.
  The thoracic (rib) cage is well developed, and the sternum bears a
  pronounced keel for the attachment of the pectoral muscles, which move
  the flippers. The flipper has the same skeletal base as the wing of
  flying birds but with its elements shortened and flattened, producing
  a relatively rigid limb covered with very short feathers—an ideal
  organ for rapid propulsion. The body plumage likewise consists of very
  short feathers, which minimize friction and turbulence. The density of
  the plumage and the layer of air that it retains provide almost
  complete insulation of the body.

https://www.britannica.com/animal/penguin#ref3467


Can systemic venous congestion caused by tumor? I am thinking, if 1) little compressed superior caval vena and 2) some vein compression by tumor, can cause systemic venous congestion.

I think this is only in rare occasions.
However, the question is if this is possible.

Can systemic venous congestion caused by these two factors?

No. They increase the risk, but are not the causes.
There must be other factors involved which triggers the systemic venous congestion.


Why can't dopamine cross the blood brain barrier but l-dopa can? Levodopa can cross the BBB and is less lipid soluble then dopamine, so dopamine should be able to cross the BBB more easily then l-dopa, but for some reason dopamine cant cross the BBB. Both dl-methamphetamine and levomethamphetamine can cross the blood brain barrier, but why not dl-dopamine?

Both L-Dopa and Dopamine are polar molecules and thus hydrophilic. They are solvable in water but not in lipids, so they are not able to cross the Blood-brain barrier on their own.

The difference is that L-Dopa has a transport protein (SLC7A5) that allows it to pass through the Blood-brain barrier, whereas dopamine doesn't.


Why does menthol clear a stuffy nose? I've looked all around online for an answer to this. Some sites say menthol causes blood vessels in the nose to constrict, some sites say it causes blood vessels to expand. Many sites simply say it's a remedy, but with no explanation.

And if it does cause the constriction or expansion of blood vessels, what's that got to do with making the stuffiness go away?

Menthol is stimulating action potential at cells which are inducing thermoception. As far as I have found out it does not have any effect beyond that. The patients only feel like they can breath better while it could not be proven that they actual do.

https://www.ncbi.nlm.nih.gov/m/pubmed/18702906/

The blood vessel thing : In general "cold" is a cause for vasoconstriction - that might lead to the conclusion that menthol is doing the same. I have no experental background on that but - after online research :-D- it seems like that menthol causes vasodilatation.


Can you find malaria parasites in saliva? Can you find plasmodium parasites (malaria) in saliva under microscope from someone who's infected? Or it's only in the blood?

No.
Although malaria is transmitted through the saliva of a female Anopheles mosquito, it stays in the bloodstream and doesn't pass over to the saliva of humans (otherwise it probably would be transmittable via humans directly). Once the parasites travel to the liver, it infects and bursts hepatocytes after reproducing (asexually). The burst cells then infect the bloodstream via red blood cells, where the parasites reproduce further and eventually burst their host cell, leading to a vicious cycle. This whole process only has to do with the bloodstream, since the parasites live in red blood cells.

However, there are certain markers present in the saliva which can be used to detect the presence of malaria the most known of which is called PfHRP2, or Plasmodium falciparum histadine-rich protein 2. This only applies to people infected by the type of parasite P. falciparum, however it is this parasite type which is the cause for the vast overwhelming majority of deaths and complications during malaria infection. The range of PfHRP2 values in malaria-positive patients' saliva ranged from 17 to 1167 pg/mL in one study.

References:

http://www.malariajournal.com/content/11/1/175
http://www.jstor.org/discover/10.2307/30086299?uid=3738432&uid=2&uid=4&sid=21104875852967


Except for protecting ears from cold air, does a woollen cap provide any other significant advantage in winters? I have woollen earmuffs and they are really comfy. They do the job of protecting ears well enough and I don't really have to worry about hair statics as they only cover ears and not my hairs. But many times I do get advice against using those earmuffs and wear a woollen cap instead.
If protecting ears is the only reason of wearing caps than I don't think i should leave my earmuffs but most people, though unknown of any scientific proof, claim that caps have more of a advantage than just that. Is it really so?

This article is what you need: http://www.bbc.com/future/story/20130708-is-most-heat-lost-from-your-head . Citing an excerpt from it:

[H]aving your head immersed in cold water only adds 10% to your overall heat loss in a cold pool. And given that the head accounts for 7% to 9% of the body’s surface, that doesn’t seem excessive.
But before you throw your collection of woolly hats in the bin, there’s another finding to bear in mind: when the head is allowed to get cold and the body is effectively insulated, the body’s core temperature drops a lot more rapidly than most people would expect.

In the end, wearing a woollen cap prevents the body's core temperature to drop too low. So if the rest of your body is well covered, you can skip the cap and put just your comfy headphones on; if, on the other hand, your clothes don't protect you well, putting a woollen hat on may be the easiest way to not feel cold.


Is Araucaria fruit toxic? I've had an Araucaria tree in my garden for more than 30 years.
Today a fruit fell off the tree. It looks just like this one:

I want to know if it is toxic?

Can I eat it? If so, how?

Araucaria araucana is an evergreen. The pictured "fruit" is actually more of a cone (which I don't think you'd want to eat). However, the seeds are edible, and look like this:

Let the cone dry and it should open up.


Blood pressure measurement When measuring your blood pressure in your arm, when you hear the sound to measure the systolic, is that sound NOT the heartbeat?

The sound in question is called the Korotkoff sound:

  If the pressure is dropped to a level equal to that of the patient's
  systolic blood pressure, the first Korotkoff sound will be heard. As
  the pressure in the cuff is the same as the pressure produced by the
  heart, some blood will be able to pass through the upper arm when the
  pressure in the artery rises during systole. This blood flows in
  spurts as the pressure in the artery rises above the pressure in the
  cuff and then drops back down beyond the cuffed region, resulting in
  turbulence that produces an audible sound.


How do neurons send electric pulses even though they are surrounded by liquid which is mostly water? I just thought about the fact that people move through electrical pulses traveling through neurons in the brain and muscles through the body. Here is what i know: the body is full of 97 or more, percent of water. the thoughts we have and the moves we make are a result of electrical impulses within the neurons and cells.
  My question is why the water does not disperse the impulse and or make it completely ineffective. My personal guess would be that each neuron is made of or has an outer layer of electric resistance material that( is it conducts) electricity.

Neurons have sodium, potassium, as well as some calcium channels all over the surface of the cell (I'm giving a very generalized explanation here). The "electric pulses" that you are mentioning is more of electrostatic potentials generated from the interactions of cations and anions flowing in and out of the channels. Water at the specific pH surrounding the cations is a good conductor of these electrostatic interactions.

You have to remember that there are large negatively charged anion proteins inside the neurons which attract the cations (sodium ions for example) to rush into the cell. Once threshold is reached all gates open and an action potential is produced.


Branch taking over a tree trunk I stumbled upon a birch growing in sandy soil in a coniferous forest in central Russia.
It looks like over time the tree trunk got bent towards the trail and one of the branches became the new trunk as it now grows straight up, whereas the old trunk is pointing sideways.1

The tree isn't dead, I visited that place during summer time and it was covered with green foliage.

I'm wondering what's the name of such phenomena, how common it is and what usually causes the tree "to change it's mind"?

1. I apologize in advance if the terminology I'm using is inaccurate, so feel free to edit my question.

This phenomena has elements of what,  in the UK, are termed Phoenix Trees, however this term, and the term Phoenix Regeneration, is more often used when describing characteristics of ancient and veteran trees.

The principles that underlie what the photo shows is that new growth on trees will always grow strongest where there is the most light available, and so in this case as the trunk has become bent towards the canopies, and the shade, of neighbouring trees this branch has become the part of the tree to encounter the most available light.

It is possible to find many trees in a woodland that may show elements of this phenomena; your photo is of a tree that is on the more extreme end of the spectrum for a tree that is still standing. It has developed a different 'dominant' stem to the one it 'started' with.

It is probably not now possible to discover what has happened in the past to cause this tree to bend over in this way; it could have been perhaps a heavy build up of snow or ice at a particularly vulnerable point in its growth, or more likely one or more other trees, or a part of another tree perhaps falling in such a way to knock into it and stay in that position long enough for this tree to adopt this shape in its trunk. If this was the case it may be that the tree/s that fell into it has since been cleared, or decayed away if it was a dead tree or branch.

Often trees that are completely blown over can remain alive because enough roots remain in the soil for it to survive. When this happens the branches on the trunk start to grow upwards into the new space and light that has been created by the tree itself falling. Over time the result is a tree where the main trunk is lying on the ground but the canopy is now a series of branches at right angles heading straight up.

The name for a tree that continues to live and grow after it has fallen over is a "Phoenix tree", named after the mythical bird that regenerates itself after catching on fire; it rises from the ashes.

This term is often applied to old trees, but it could, and does, also be used to describe any tree that has fallen over but continued to grow.

References can be found in Natural England's excellent publication on Veteran and Ancient Tree management
http://publications.naturalengland.org.uk/publication/75035

For example in Chapter 2 "What are Veteran Trees and why are they important?" Section 2.3.4.6 (page 21) of Veteran Trees: A guide to good management

Old trees may fall over completely and then re-grow or collapse and layer well away
from the original base. This is characteristic of lime, willow, alder, black poplar, medlar
and bird cherry but can occur in any species. A particularly notable example is the
Tortworth Chestnut.

The term phoenix regeneration has been applied to trees that have fallen over, or
split apart, and successfully continued growing.


What causes the sound of climaxing bees? I just saw the funniest video of bumblebees(?) having sex. (mirror) As they were getting it on, there seemed to be an almost audible 'moaning' coming from the pair. From what I presume it was a sexual climax we heard!
Bees have neither vocal chords nor lungs, so I wonder: what is that sound?

As the pair is mating on a newspaper, I'm almost certain we're hearing transduced vibrations from the bees made audible through the paper and not the bees themselves. (Nonetheless, what a beautifully humorous anthropomorphism! Moaning bees certainly give vivid color to the birds and the bees!)
I have multiple theories:

The ejaculation of sperm by the drone is known to be 'explosive', even rupturing the endophallus and making a 'popping' sound. I wouldn't call the sound in the video 'popping' by any means, though.
(Un)controlled movements of legs or wings by either of the pair. This seems very likely considering the subjective frequency of the sound, but that's just speculation on my part.
Perhaps it's the queen piping? I'm really grasping with this one. I don't even know if that species' queens pipe or even have the ability to pipe.

Any excited entomologists wanna fill me in?

Firstly, those aren't bumblebee faces ... those are big fly eyes. Those are Bumblebee Hover Flies (Volucella bombylans):

The noise in the first part of the video is identical to a fly flying around your head and the noise at the end sounds like what a fly would sound like when you press it against a surface like a wall or screen (or a newspaper, apparently)


What evolutionary reason is there for having the urinary duct and reproductive organs so close together? As the old joke goes, "God must have been a civil engineer. Who else would put a waste facility straight through a recreational area?"

But maybe it wasn't God. Is there any evolutionary reason (or background for) having the urinary duct and reproductive organs right next to each other (in both humans and many other vertebrates)?

To be clear, I'm not asking why isn't it elsewhere. I'm asking where the original "design" came from that spread everywhere.

The reason lies within our wormy chordate ancestors - an orifice used to eliminate waste can also function as an orifice to eject eggs. My vertebrate zoology is a little bit rusty so I would suggest picking up any first-course book on the subject, most of them cover the evolution of urinary/reproductive organs extensively.


Are there proteins that stabilize mRNA:RNA polymerase or mRNA:ribosome complex? Actinomycetes are known for their ability to produce rich variety of natural products, and particularly, polyketides. Many of the genes that encode the biosynthetic pathways are pretty big, as they could reach 20kb. These bacteria most probably have some kind of mechanism that would ensure efficient transcription and translation of these long ORFs. This could be done in two ways: either by stablizing the mRNA:RNA polymerase complex, or by stabilizing the mRNA:ribosome complex. Have any of you heard of such mechanisms in bacteria?

I have searched for information on this topic as I used to work in the area of protein biosynthesis, but without success. However one argument against there being a tendency for ribosomes to fall off mRNA is the very specific mechanism for ribosome release after the stop codon has been reached (described here).

There are other possible problems with very long mRNAs:

Possible degradation of (prokaryotic) mRNA during a long translation time. I would imagine that the mRNA would have to contain structural features that prolonged its life.
Possible misfolding and precipitation of the protein product before the whole protein is completed. I think there are probably chaperone proteins that protect hydrophobic regions or encourage folding of domains to prevent this.

As a footnote, the longest mRNA is thought to be that for the muscle protein, titin. The assembly of that on the sarcomere would not be typical of polyketides, say, but other problems encountered might be similar.


Have there been any measurements on the genomic differences between cancer cell DNA and healthy cell DNA? I'm loooking for simple stats like the average number of base pair differences to normal cells, average diversity between different cancer cells, etc.

Yes, there are two genome projects, which aim to identify the critical mutations (and by looking for them, they discover all the mutations in a given cancer) by sequencing DNA from the cancer cells and healthy cells from the same patient.

These are "The Cancer Genome Atlas" (TCGA) and "The Cancer Genome Project (TCGP)". Both project publish their complete data and ways to browse them. The data from the TCGP can be found in the "Catalogue of somatic mutations in cancer" (COSMIC), the data from the TCGA on their project site.


Do other animals suffer from myopia or hypermetropia? Mammals have eyes similar to humas and many other animals like octopuses have a lens in the eye. So do they have such eye defects? If yes, how do they overcome it? Do they feel selection pressure against them in such cases?

do they have such eye defects?

Yes, eye defect is not specific to humans of course. Cases of eye defect are very common in animals. It is most often witnessed in domestic animals and they are the ones we observe the most (see The physiology of domestic animals)

Eye defects have typically been observed in tree shrews, monkeys (incl. macaques), cats, dogs, horses, rats, elephant and a Cape buffalo.

You should have a look at the wikipedia article Myopia in animals

  howw do they overcome it?

They don't wear glasses obviously!

Humans uses the sense of vision a lot. Many animals uses smell (and taste), hearing or touch much more than humans do. For such non-visual animal, having a slight eye defect is not too big of a deal.

  Do they feel selection pressure against them in such cases?

That is a bit of an awkward question. Nobody can feel a selection pressure on themselves. A selection pressure is a fitness differential associated with genotypes. A selection pressure is therefore defined at the population level. There is no such thing as a fitness pressure against a single individual.

Let me rephrase this last question to something that makes more sense. I am not sure I will address the specific question that was of interest to you though.

  Are non-humans animals that have an eye defect aware that they have an eye defect?

It is unlikely that any animal would have the cognitive abilities to notice that they see more poorly than another animal of the same species. Doing so, would at least require having the ToM and only few species do.


shRNA knockdown not showing with Western I've recently been performing shRNA transfections and was hoping to get some advice on why I'm not seeing any knockdown via western blot

I transfected by MDAMB468 breast cancer cell lines with shRNA against PTEN from the pLKO.1 lentiviral vector. I selected for the transfected cells using 1ug/ml  puromycin for approximately 6 days. During this time there was very few cell death amongst the cells compared to my control plate (untransfected cells with 1ug/ml puromycin), suggesting that the transfection was successful.

After the 6 days I harvested my cells for protein and RNA extraction. I performed a western yesterday to assess knockdown, but noticed that there was hardly any differences (if any!) compared to my shGFP control. I haven't done a qpcr yet but I plan to this week.

I was wondering if anyone has experienced something similar, especially when working with PTEN? could it be that the cells need to be selected for a longer period (i.e. more than a week) before assessing protein knockdown?

Thanks!

I assume that you transfected the plasmid directly not producing viruses. If you want stable transformants, you might want to subculture them and maintain a bit longer. It is because efficiency of integration of plasmids into genome could not be that high--less than 0.1% of population obtain stable resistance by integration into genome. After 6 days, cells were still healthy in your case. This means you might get good transfection efficiency and after several times of cell division, your plasmids were diluted and the effects might be low even if cells maintain resistance against puromycin.

If transient expression (KD) works in your experiment, you might want to check expression level of your target at day 2 or 3. If you prefer stable cell lines, you can increase puromycin concentration, and if you agree, you pick up single colonies after cells not having plasmids in genome are dead. Among these colonies, some have more plasmid copies other have less. You can choose cells least expressing your targets as well as moderately expressing cells if you want. Because you might want to see expression level dependency.

For some cells, 5ug/ml puromycine is used according to this link.


Antigen Antibody reactions during blood donation A person wih blood group O is called a Universal Donor. Well, his plasma contains antibodies A and B. During blood donation, if blood group O is given to a person with blood group A (since blood group O can be donated to all the blood groups) then wouldn't the antibodies of the donor with blood group O harm the recipient?
Although nowadays doctors prefer giving blood having 100 percent compatibility...

The key feature of type O blood as "universal donor" is that the incoming red blood cells have neither A nor B antigens and so the resident antibodies (anti-A , anti-B) will not react with them. Since transfusions are carried out with packed red blood cells the plasma antibodies of the donor don't matter - they will not be introduced into the recipient.


How long can the brain survive during ongoing cardiac arrest? There was this interesting discussion on CPR and defib in response to the question "Why can't we defibrillate the heart within 1 minute after ventricular fibrillation by electroshock?".

Now I was wondering "How long can the brain cope without O2?".

And to specify this question to a realistic situation: "How long can the brain survive a cardiac arrest without (gross) neurological symptomatic consequences?"

I would define "gross symptomatic consequences" as "noticeable and debilitating" in one way or another to the subject. This opposed to minute changes in, e.g., motor response latencies measured in a laboratory task designed to pick up the slightest results of asphyxia.

As background: I did these CPR and defib courses and the first thing they tell you to do is (1) call 911 / 000 whatever alarm number and (2) arrange a partner in crime to help with CPR;  or in case of a defib situation, (call alarm nr), make your way to the defib, unpack, apply electrodes, look at the manual, casually wait for instructions/warning lights etc. But..but... there is a person here without oxygen supply to his brain! And I know professional staff should be brought to the scene, but what struck me most is that starting up the defib (which inherently takes time) is more important than CPR (which is immediate)? In other words, loosing precious minutes is advantageous over diving straight away into CPR and pumping blood around?

From Boron and Boulpaep textbook of Medical Physiology, second edition, p.289:

  Because of falling ATP levels in the brain, consciousness is lost within 10 seconds of a blockade in cerebral blood flow. Irreversible nerve cell injury can occur after only 5 minutes of interrupted blood flow.

If conscious is lost within 10 seconds of blockade in cerebral blood flow, I guess a person will not be conscious to do any action in only 10 seconds!

Brain ischemia is divided into

Focal ischemia which signifies stroke.
Global ischemia which occurs in cardiac arrest.

Permanent neural injury means that once perfusion is restored, the symptoms don't relieve as the part of brain which caused those symptoms becomes dead irreversibly.

For a list of symptoms check this page.


Can our body adapt to periodic fluctuations in sleep? Suppose that if I've to attend a morning class then I must wake at 5 AM in morning. Next, if I don't (attend the class) then I wake up at 7-8 AM.

I've experienced that, since I do attend those classes almost regularly, my body has adjusted itself (sleep cycle) to wake at 5 AM almost precisely without a alarm set and has also decided when to sleep (it seems to demand 7-8 hour of sleep). This, I think, is common to most of us.

Now suppose that I attend classes only on alternate days. Put simply, I wake up at 5 on first and at 8 on the next day and so on. This implies that I am constantly switching my sleep cycle every day. Would my body, now, be able to adapt to this continuously varying cycle (which is but periodic).

I suppose no, and that is just a guess based on my experience. But provided I give my body enough time, I think that it may really adjust to this scenario.

On a side note, assume that (in case it matters) I take a sleep of 7 hours during class days and a sleep of 8.5 hours during holidays to make this data realistic. .

In the same way that it takes time to fully adjust to jet lag (~ 1 timezone/day), it is unlikely that the body can adjust to alternating patterns of 3hr gaps on wake time on a daily basis [Ref. 1].  We simply have not evolved to jump timezones.  The most healthy thing to do is to have one time to get up each day (5am) so that your body is in sync.

[Ref. 1]: Review about circadian rhythms