Designing genes with DNAWorks: Maximum nonzero score?

You might want to read Gibson's paper on the step-wise assembly of the mouse mitochondrial genome (1):

He started with 60b long oligos with 20b overlap, as he assembled 5 of those 60-mers into a backbone, obtaining 384b fragments. On the next step, he joined 5 of those 384mers, obtaining 1.2kb constructs. You can do the same, but on the second step use 2x 384mers to get you ~600bp gene. Gibson didn't use DNAWorks to chop up the sequence. He just started from base 1, so that his fragments were F1 [1:60], F2[41:100], F3[81:140], etc.

I think that 15b homology is pretty low (Gibson used 20b for assembling the 60mers in the first step, and 40bp homology for the next assemblies). 60b oligo length is standard.

You can also try one step assembly with all of your oligos (via PCA) as you planned, but I think that the two step assembly might be more efficient.

Let me know if you need more help.

1. Gibson et al, 2009. Chemical synthesis of the mouse mitochondrial genome


No.
But close.  Very close.  Basically, a gametophyte is a spore that has / is undergoing mitotic division.  Therefore, it is still haploid, having not undergone fertilization yet, but it is no longer a dormant, unicellular spore; it's passed into the new phase of gametophyte.
The gametophyte produces sexual organs which produce the gametes that will actually be used in fertilization.  Two gametes fuse, at which point, a diploid cell is produced (called a zygote, just as in animals).

The meiospores are the spores you see in the picture on the far left: they are dormant, unicellular, haploid spores, which will turn into the gametophytes.  (meio + spores; spores that have undergone meiosis)

Sources:

Meiospore, Biology Online
Gametophyte, Wikipedia
Plant Life Cycles, Developmental Biology, 6th ed.
Spore, Wikipedia


Why does hemoglobin electrophoresis in sickle cell trait gives two bands but not three?

This is a really interesting question and again reminds of how ignorant we are about things as subtle as this which otherwise should have been an obvious question.

First of all, although there seems to be no confusion, I'd like to clarify that we are talking of the protein being separated on electrophoresis. Hemoglobin electrophoresis classically refers to the cellulose acetate/citrate agar electrophoresis where the RBC hemolysate is used as the sample. This method uses the fact that, hemoglobinopathies generally have mutated Hb which differ in charge from the original HbA. Charge is a property of separation in cellulose acetate electrophoresis unlike SDS-PAGE where separation is on the basis of mol. wt.

We can also do a DNA electrophoresis (digressing) to identify a Hbpathy after digesting with restriction enzymes which would give a different results, depending on the restriction enzyme that has been used to differentiate the mutation from the original. Here is an example using MstII for RFLPs. This isn't the topic of discussion here. Our sample is the protein, and not the DNA.

So it is important that the electrophoresis we are going to talk of, is of a protein electrophoresis based on charge.

Considering the sickle cell trait, let us clarify the terminology now. HbA is normal hemoglobin tetramer which composes of $\ \alpha_2\beta_2^A $. HbS on the other hand is $\ \alpha_2\beta_2^S $. The terminology you have used not only isn't standard but also is conveying a different meaning. Now logicaly, in a person with sickle cell trait, one should be expecting a hemoglobin with both different beta chains. And it does happen! Literature calls it the assymetric hybrid. Let us indicate it with AH which is $\ \alpha_2\beta^A\beta^S $. Let's sum this up a bit.

HbA = $\ \alpha_2\beta_2^A $

HbS = $\ \alpha_2\beta_2^S $

AH = $\ \alpha_2\beta^A\beta^S $

The assymetric hybrid that has been mentioned here is only for the sickle cell trait. There are many other hemoglobinopathies and in their heterozygous states one can obtain other assymetric hybrids with a different beta chain.

Now that we know that assymetric hybrids are formed, why do they not show up on the Hb electrophoresis?

It is generally believed that under physiological conditions, at equilibrium, hemoglobin is a mixture of tetramers ($\ \alpha_2\beta_2$) and dimers ($\ \alpha\beta $) as $$\ \alpha_2\beta_2 ⇌ 2\alpha\beta $$ The dissociation equilibrium constant for oxyhemoglobin is approximately $\ 2x10^{-6} M $ (heme) and is much less for deoxyhemoglobin. The asymmetric hybrid is generally not isolated by conventional techniques, since the tetramer-dimer dissociation constant is much lower in-vitro than in-vivo . Thus the asymmetric hybrid dissociates into dimers $\ \alpha\beta^A:\alpha\beta^S$ and reassociates into more symmetric forms during the separation process.

There are slightly modified methods to isolate the assymetric hybrids under deoxygenated conditions such as isoelectric gel focussing, cross linking the chains, etc. The amount of AH formed in proportion to HbA and HbS can be measured in these kind of experiments and has been found to be comparable to the expected binomial distribution of $\ a^2 + 2ab + b^2 $. This implies that the stability of the AH is comparable to HbA and HbS.

Hope it helped.

To delve further, here are a couple of reference that should be of use.

  HF Bunn and M McDonough (1974), Assymetrical Hemoglobin Hybrids.
  An Approach to the Study of Subunit Interactions, Biochemistry,
  13(5)989-93.
  MK. McCormack, TG. Westbrook, RM. Paull & S Berman (1981),
  Identification of the Asymmetric Hybrid of Human Oxy Hemoglobins A
  and S (α2βAβS) at 4° C by Cellulose Acetate Electrophoresis,
  Hemoglobin, 5(3)251-56.


What is the mechanism of cytokine release syndrome in CAR T cells?

CRS in CAR T therapy is likely the result of hyperactivation of a large number of immune cells, either by antigen-specific/co-stimulatory activation (T cells) or by non-specific activation due to significantly elevated pro-inflammatory cytokines. CRS is the most prevalent adverse effect following CAR T infusion, but does not always happen. These two reviews provide a lot of detail, and reference many primary studies, but I will summarize briefly below:

1) http://www.nature.com/articles/mto201611

2) http://www.bloodjournal.org/content/124/2/188?sso-checked=true

In patients with CRS, it manifests clinically whenever maximal activation of the infused T cells occurs. From ref 1,

"In the setting of early CAR T-cell trials utilizing “first-generation” constructs (without costimulatory signaling elements), insufficient T-cell proliferation/cytokine production and lack of antitumor response were noted.30 The addition of costimulatory signaling in second-generation CAR design (CD28 or 41BB) translated to improved T-cell activation/expansion, cytokine production, and most notably dramatic antitumor responses in patients with hematologic malignancies.3,31,32"

So the 2nd-gen CAR design leads to better antitumor responses, but can also cause an intense burst of cytokine release that sets off a sort of 'chain reaction' wherein many other native immune cells also begin to massively release cytokines. One of the key pathogenic cytokines in this cascade appears to be IL6. From ref 2,

"Emerging evidence implicates IL-6 as a central mediator of toxicity in CRS...[]... IL-6R is cell associated on macrophages, neutrophils, hepatocytes, and some T cells..[]... High levels of IL-6, present in the context of CRS, likely initiates a proinflammatory IL-6-mediated signaling cascade"

and from ref 1,

"As an effective alternative IL-6 receptor (IL-6R) blockade with the Food and Drug Administration-approved mAb, tocilizumab has demonstrated near-immediate reversal of CRS.3,35 Investigators are now determining the effect of IL-6R blockade on CAR T-cell proliferation, persistence, and most importantly, antitumor effect. Despite this unknown, the use of IL-6R blockade has generally been accepted as front-line treatment for sCRS following CAR T-cell infusion.23,31,36"

Finally, it does appear that tumor burden has been correlated with CRS severity, presumably because of the higher antigen load available to activate the CAR T cells. From ref 1 and 2, respectively,

" It has been demonstrated that the degree of CRS severity is dictated by disease burden at the time of infusion as patients with high tumor burden experience a more sCRS.3,31,32"

"In recent reports of CRS following adoptive T-cell therapy for cancer,10,14,8,28 the incidence and severity of the syndrome also appears greater when patients have large tumor burdens, presumably because this leads higher levels of T-cell activation."

Hope this helps. Clearly there is a long way to go in terms of predicting and managing these adverse events.


Will a person with two copies of the same set of chromosomes be normal?

It sounds like this person would be close to what you get with "pure lines", aka inbred strains in laboratory animals:

  A population of identical homozygotes constitutes a pure line. Pure lines "breed true"; that is, they maintain their phenotype indefinitely if genetic crosses are restricted to members of the pure line (see True Breeding).

You get those by inbreeding for enough generations that the individuals become genetically identical, or monozygous for every allele at least.

And on the effect of being a homozygote for all genes:

  Inbreeding in allogamic organisms bring the deleterious recessive alleles to homozygosity; the immediate consequence is an increase in the frequency of defective offspring, or, in another words, an increase in the genetic load of the population. This phenomenon is called inbreeding depression or inbreeding degeneration. As inbreeding continues, the deleterious alleles are selected out and eventually disappear.  The original heterozygous populations are often more fit than the resulting pure lines because they profit from heterosis and balanced polymorphisms; the main advantage of pure lines is the quick production of many individuals with the same well-adapted genotype

It sounds like she would only have genetic problems if she had problem genes; bad alleles are more likely to be harmful if you're monozygotic for them because there are no other alleles to compensate, but if you only have the good alleles to begin with there's no harm. You would have problems in cases where having two different alleles is itself superior to having a single one of any; for things like immunity for example that can be the case. Or being heterozygous for the sickle-cell allele in malaria country, for another standard example.

I don't know if any of those things would be harmful enough that we'd call that woman "unhealthy" or "suffering from genetic problems" though. I think if it did, it would be more a statement about how the human genomes contains these specific alleles that you have to be heterozygous for than about the intrinsic harm of being the combination of two identical haploid genomes.

As for whether her egg cells would all be identical, they should be since homologous recombination should be switching identical bits of DNA with each other, but that's ignoring mutations. Which shouldn't be ignored, because every human has on the order of 100 mutations that weren't in their parents. Still, compared to how much DNA there is in the eggs overall it would also be reasonable to call them identical.


During starvation, does the human body do anything to prioritize which organs receive nutrients?

The glycogen in the liver begins providing blood glucose. Muscle glycogen is used as fuel by the muscles, fat cells (adipose tissue) release fatty acids to manufacture ketone bodies in the liver and to be used by the brain as fuel, and body proteins are converted to glucose.

In short, the body's metabolism shifts to catabolic reactions.

If this continues for too long, you begin to see effects of starvation:

Inadequate tear production
Enlarged/tender liver
Muscle atrophy
Rashes, sores, or peeling skin

In short, deficiencies of energy, protein, iron, and zinc can really mess you up in the long term.


Why don't vegetables taste good despite being healthful?

We are evolved to survive starvation, and live to be perhaps 35. So fatty foods with lots of calories taste good to us. Our genes (and preferences) lag thousands of years behind our present environment.


Specificity and sensitivity are terms used in the pharmaceutical and in vitro diagnostics (IVD) industries that have specific meanings relating to how the particular assay in question was validated. Generally the values are being compared to spiked standards in matrix using recombinant protein or purified virus or whatever the antigen is (depending on how the test operates) where the analyte concentrations are known. With a certain number of titrations and assay repeats, along with a bunch of statistical calculations, you can then compute your expected and actual recovery, as well as values like the LLOQ (lower limit of quantitation) which gives you your sensitivity (we can reliably detect 1e4 viral particles per ml, for example).

Specificity is calculated using related antigens, such as proteins from other types of bacteria that are of varying degrees of relation to syphilis, or perhaps of different serotypes that aren't claimed to be detected by the test, or whatever. Again, you spike known amounts of these materials into your testing matrix (blood, sputum, urine, semen, whatever) and see what your test returns. So, say I have a virus with 2 serotypes (A and B) that I claim recognizes A specifically. I spike in perhaps equal amounts (for example, 100 units) of A and B, then run the A-specific quantitation assay. The readout might then say the sample only had 75 units of A, meaning 75% specificity of the reagents.

For more reading, search for "clinical assay validation" or something similar. There are a ton of papers out there giving guidance for particular types of assays, as well as regulatory agencies' interpretations of the relevant laws covering the tests. There are also many books, reviews, etc. if you're just looking for an overview. It's a huge field, and I've just given a very basic description to avoid writing pages and pages.


How much gas is exchanged in one human breath?

According to Wikipedia

  "In a healthy, young adult, tidal volume is approximately 500 ml per inspiration..."

(tidal volume is the volume inspired/expired)

Using this figure, together with values for gas composition also taken from Wikipedia, I estimate that in each breath we take in 18 mg O2 (1.1 mmol) and we release 36 mg of CO2 (1.2 mmol) plus 20 mg H2O (1.1 mmol). These are, as you say, ballpark figures.

Sample calculation:

O2 inspired = 21% by volume; O2 expired = 16% by volume

O2 change = 5% by volume = 5*500/100 = 25 mL

1 mole gas = 22.4 L; 1 mmol gas = 22.4 mL

O2 change = 25/22.4 mmol = 1.1 mmol

MW O2 = 16

O2 change = 17.6 mg

The relative values are reassuringly close to what you might predict from the textbook equation for oxidation of carbohydrate: C6H12O6 + 6O2 -> 6CO2 + 6H2O


The distinction between male and female is not that clear in plants, at least at the level of individuals. Actually the details may be rather complicated (see [1]), but in general we can distinguish between plants that have:

male and female individuals (dioecy)
male and female flowers on the same individual (monoecy)
flowers that have both male and female structures (bisexuality)

In the first case, only female individuals produce flowers that -- when fertilized by the pollen from the flowers of a male individual -- develop into a fruit. Examples are Hazelnut, common nettle and a few other species [2]. However, dioecy is rather rare among extant flowering plants [3].

Monoecious plant species are somewhat different. A good example is maize. The male flowers of a maize plant are located in the tassels at the apex of the stem, wheres the female flowers are are in the leaf axils. Note that each corn developed from a single flower and that the cob is actually the infructescense.

Then there are bisexual plants. In these, every flower has the potential to be fertilized and develop into a fruit, which is why they are also called perfect flowers. Plants have developed several strategies to prevent self-fertilization, e.g. the temporally delayed expression of female/male structures within the same flower.

Resources:

[3]: Bawa, K. S. (1980). Evolution of dioecy in flowering plants. Annual review of ecology and systematics, 15-39.


Species Identification : Worm like insect but broad at back

That actually looks like some sort of caddisfly larvae in its case to me.

Are you sure that's it's 'body' & not a case?

Did you find it in water?

Or a Case Bearing Clothes Moth (Caddisfly are closely related to moths).

This one (a clothes moth larvae) looks very similar to your image.


Why is a solution of cesium chloride used in Meselson & Stahl's DNA replication experiment?

In saline solution all the DNA ends up at the very bottom.
Under centrifugation, the Caesium Chloride solution forms a density gradient, each DNA rises or sinks to the equivalent density.
The same procedure is used with Sucrose solutions for other separations.


Do plants with non-green leaves have chlorophyll and photosynthesis?

Yes, it is possible, but not necessarily the case.

Non-green leaves with chlorophyl: There are leaves that don't appear green, but do have chlorophyl and therefore can conduct photosynthesis. (See, for instance, refraction effects in white caladiums or the link in the answer by Resonating).

Non-green leaves without chlorophyl: There are leaves that don't appear green because they have no chlorophyl, and so do not conduct photosynthesis. (See, for instance, parasitic plants like the Oronbanche linked by Cactuswoman or this Monotropa)


Why does uncompetitive inhibition decrease the Michaelis constant?

First off, the difference between the types of inhibition:

competitive inhibition: The inhibitor only binds to the substrate-free form of the enzyme. (Not necessarily at the active site!)
uncompetitive inhibition: The inhibitor only binds to the substrate-bound form of the enzyme.
noncompetitive inhibition: The inhibitor binds equally well to both the substrate-bound and substrate-free forms of the enzyme. (Some people feel this is just a non-special special case of mixed inhibition.)
mixed inhibition: The inhibitor binds to both the substrate-free and substrate bound forms of the enzyme. (But not necessarily with the same affinity.)

Secondly, you need to separate out the concept of binding affinity of the substrate from the concept of $K_m$. The binding affinity of the enzyme towards the substrate is just what you think it is: the equilibrium constant between the bound and unbound forms of the enzyme, in the absence of catalysis and inhibitor.

The $K_m$, on the other hand, is an overall parameter of the rate equation, and doesn't necessarily have a direct relation to substrate binding. The confusion between the two arises from the fact that in simple enzymatic systems, under the simplified conditions we normally study them in, $K_m$ is a pretty decent approximation of the substrate binding affinity. Thus you'll have introductory textbooks saying that "$K_m$ is a measure of substrate affinity". -- a gross oversimplification. They're certainly related, but they're not exactly identical - especially when you get to more complex reaction schemes. Confusing the two leads to problems.

The enzyme inhibition cases are an example of this. Enzyme inhibitors (even competitive ones ones) don't change the intrinsic binding affinity of the free enzyme to substrate. Instead, they draw off some of the enzyme from the productive pathway. Because they change the concentration of particular enzyme species, they change the observed rate equation. How they change the rate equation depends on which enzyme species they change the concentration of.

For a full accounting, you really should look at the derivations of the rate equations, and how the extra equilibria of the inhibitor binding changes things. (e.g. here or here) But for something of an intuitive explanation:

pure noncompetitive inhibition reduces the concentrations of substrate-bound and substrate-free forms equally. (The concentration of product-bound forms being negligible in the standard initial rate equations.) This is basically the same as reducing the total amount of enzyme, i.e. reducing $V_{max}$ or apparent $k_{cat}$.

competitive At saturating substrate concentration, the amount of free enzyme (and thus the effect of inhibition) is negligible. The rate at saturating substrate (i.e. $k_{cat}$) is thus the same. Therefore, it's the apparent $K_m$ that changes. (NOTE! The actual affinity to the substrate isn't changed by the inhibitor. Instead, the inhibitor specifically reduces the amount of free enzyme available, in a way that can be compensated for by higher substrate concentrations. This just so happens to change the rate equation in the same way changing the $K_m$ does.)

uncompetitive At negligible substrate concentrations, the amount of substrate bound enzyme (and the effect of inhibition) is negligible. Thus the rate should be the same with and without the inhibitor. If you recall, the enzymatic reaction rate at negligible substrate is parameterized by $k_{cat}/K_m$. For uncompetitive inhibition, this needs to stay the same. Thus both $k_{cat}$ and $K_m$ need to change in the rate equation, to the same proportion.

mixed - A mixture of the above effects, depending on the relative affinities to the substrate bound/unbound forms of the enzyme.


Why does flu vaccination only work against specific strains?

I'm going to try and focus on the latter part of your question; if you want to know why the tri- or quadrivalent flu vaccine doesn't provide protection like many other vaccines, that's already been answered.

Anyway, the main reason we can't is because there are simply too many.  You say 187 (not sure why, 18*11=198) but the truth is that not all H1s are the same.  The different subtypes constantly mutate, so one H1N1 may not be the same as another H1N1.  There are hundreds upon hundreds of flu types, constantly changing.  And that's not even accounting for type B viruses.

When you make a vaccine, you need a certain amount of protein.  The major quadrivalent vaccine study used 15µg of protein of each strain.  The more protein you put in, the bigger the vaccine, the more it hurts, and the more likely the body is to have a significant negative reaction.  You'd have to test all of them as well - the FDA doesn't usually work on a "three is safe, four is safe, so hundreds is safe" principle.

Plus, you have to MAKE all that protein.  We're not very good at making enough for the tri- or quadrivalent vaccines now, much less hundreds or thousands of times more.  We simply don't have the capacity for that level of production.  Even if we could, it's not likely that immunity to all strains would take.  Besides, erradication is unlikely because there are large animal flu reservoirs (avian and swine, most famously) that can and will jump over to humans.  That's the piece everyone forgets about smallpox - with no animal reservoir, it was easy to eradicate.

That's not to say the cause is lost.  Intense research has begun on universal flu antibodies, and it looks somewhat promising.


confusion regarding degrees of flexion and a goniometer

There are two criteria to differ between flexion and extension:

1) Decreasing or increasing the angle between the involved bones. By this criteria, it should be clear that flexion means bending of the elbows and knees and lateral bending of the spine (the neck and torso) toward the bent side.

2. The anterior/posterior direction of the movement. All anterior (ventral) movements in the neck or torso part of the spine, shoulder, elbow, wrist, fingers, hip, ankle and toes are called flexion and all posterior (dorsal) movements (except in the knee) are called extension.

You should not use the both criteria at the same time, because they can contradict each other.

There are some specifics:

In the wrist, you can have palmar flexion, dorsiflexion (extension), ulnar flexion (abduction) and radial flexion (adduction) (Teachmeanatomy).
In the ankle, you can have plantar flexion, dorsiflexion (extension), inversion (inward rotation, adduction) and eversion (outward rotation, abduction). (ScienceDirect).
In the shoulder and hip, raising a limb to the same side as the limb is, is abduction (lateral extension) and raising it to the opposite side is adduction.
Moving the thumb toward the palm (in the same plane as palm) is flexion (adduction) and moving it away from it is extension (abduction).

You can read about flexion and extension and other movements here: Types of Body Movements (BCcampus)

  Flexion and extension are movements that take place within the
  sagittal plane and involve anterior or posterior movements of the body
  or limbs. For the vertebral column, flexion (anterior flexion) is an
  anterior (forward) bending of the neck or body, while extension
  involves a posterior-directed motion, such as straightening from a
  flexed position or bending backward. Lateral flexion is the bending of
  the neck or body toward the right or left side.

  In the limbs, flexion decreases the angle between the bones (bending
  of the joint), while extension increases the angle and straightens the
  joint. For the upper limb, all anterior-going motions are flexion
  and all posterior-going motions are extension. These include
  anterior-posterior movements of the arm at the shoulder, the forearm
  at the elbow, the hand at the wrist, and the fingers at the
  metacarpophalangeal and interphalangeal joints. For the thumb,
  extension moves the thumb away from the palm of the hand, within the
  same plane as the palm, while flexion brings the thumb back against
  the index finger or into the palm. These motions take place at the
  first carpometacarpal joint. In the lower limb, bringing the thigh
  forward and upward is flexion at the hip joint, while any
  posterior-going motion of the thigh is extension. Note that
  extension of the thigh beyond the anatomical (standing) position is
  greatly limited by the ligaments that support the hip joint. Knee
  flexion is the bending of the knee to bring the foot toward the
  posterior thigh, and extension is the straightening of the knee.
  Flexion and extension movements are seen at the hinge, condyloid,
  saddle, and ball-and-socket joints of the limbs (see Figure 1).

Now, to answer the question about the goniometer.

Again, the confusion about interpretation of this goniometer arises from two different criteria about flexion. The one mentioned in the Wikipedia and other anatomical articles says that in flexion the angle between the involved bones decreases. When you raise your arm, this criteria does not aply because the angle between the bones (humerus/scapula) in the shoulder joint does not really change (unlike in the elbow, for example), so here the criteria that all anterior movements are considered flexion applies. When you have an arm straight down, there is no flexion, so it's considered zero degrees and when it's straight up, it's considered 180 degrees, so the angle is increasing, but this is not an angle between the arm and other bones or the body but between the starting and ending position of the arm.


What virus transforms full grown plants?

Viruses can infect just about any living thing, so that it could infect a plant is no surprise.

Your article describes how a small virus-infected part of a plant had been cloned and consequently the entire cloned plant was infected, and it infected plants grown hydroponically in the same water. No surprise there either.

Plant viruses have been known for centuries. One of the earliest recorded descriptions of a virus-infected plant can be found in a poem written by the Japanese Empress Kōken (718–770), in which she describes a plant with yellowing leaves in summer. The plant (a Eupatorium) was described as "autumnal".

The top 10 plant viruses (in one paper) in molecular plant pathology were listed as:

  (1) Tobacco mosaic virus, (2) Tomato spotted wilt virus, (3) Tomato yellow leaf curl virus, (4) Cucumber mosaic virus, (5) Potato virus Y, (6) Cauliflower mosaic virus, (7) African cassava mosaic virus, (8) Plum pox virus, (9) Brome mosaic virus and (10) Potato virus X, with honourable mentions for viruses just missing out on the Top 10, including Citrus tristeza virus, Barley yellow dwarf virus, Potato leafroll virus and Tomato bushy stunt virus.

Some plant virologists even care enough to argue about it.

One paper describes how a plant was improved by a viral infection:

  In all cases, virus infection delayed the appearance of drought symptoms. Beet
  plants infected with CMV* also exhibited significantly improved tolerance to freezing. Metabolite profiling analysis showed an increase in several osmoprotectants and antioxidants in BMV**-infected rice and CMV-infected beet plants before and after drought stress.

*Cucumber mosaic virus
**Brome mosaic virus

Aetiology: The earliest recorded plant virus disease
Top 10 plant viruses in molecular plant pathology
The REAL Top 10 for Plant Viruses

Introduction to Plant Viruses, the Invisible Foe

Virus infection improves drought tolerance


Alkaline Phosphatase and Ligase Protocol for Cloning

DNA ligase catalyzes the formation of a bond between 3’-OH and 5’-PO4 of its nucleic acid substrate. (See Lehnman, 1974) The ligation bond will be formed when the proper ends are close together, allowing the ligase to act. Lets redraw your diagram and add the 5’ and 3’ designations to it. I think this may help clear out some points.

Regarding the alkaline phosphatase treatment with ligation, I think Dale and Greenaway said it so much better than I can. Quoting (Dale and Greenaway, 1985):

[The strategy] is to treat the cleaved vector DNA with alkaline phosphatase that removes the 5’-phosphate group from the DNA. Since the presence of a terminal 5’-phosphate is necessary for the action of DNA ligase, a dephosphorylated DNA strand will not be able to participate in a ligation reaction. Since this will apply to both ends of the linearized vector, the ends cannot be joined together again, i e , parental vector regeneration is impossible. This results in a very low frequency of transformation by the vector (since linear DNA transforms very poorly). However, the vector 3’-hydroxyl ends are available for joining (by DNA ligase) to the 5’-phosphate termini of any DNA fragments to be cloned. This will result in a circular recombinant molecules with a single-stranded nick at each end of the vector portion. These molecules usually transform as efficiently as the fully ligated product, and the nicks are repaired in vivo after transformation. Thus the phosphatase treatment of the vector molecules increases the proportion of hybrid DNA molecules in the population of transformed cells, and also prevents the formation of oligomeric forms of the vector.

References
Dale, J.W. and Greenaway, P.J. (1985) “The use of alkaline phosphatase to prevent vector regeneration” Methods Mol. Biol. 2:231-236. Link to paper
Lehnman, I.R. (1974) “DNA Ligase: Structure, Mechanism, and Function” Science 186:790-797. Link to paper


How and why are cells irradiated in human-rodent hybrid cell biology?

Human cells are irradiated with X rays (to fragment the DNA) and then fused with rodent cells. This is useful for mapping because closely linked markers are more likely to appear in the same hybrid: there is a lower probability of radiation induced breakage between the markers because they are close together.


Copper inhibits growth of organisms. See " Antimicrobial properties of copper" in Wikipedia.


Is there a difference between Luria Broth and Lysogeny Broth?

Although both names are quite common, both are wrong. In the original paper from 1951 Bertani was studying the lysogeny in E.coli, hence he called his media for this purpose "Lysogeny Broth" or LB in short. In the subsequent decades this name was transformed to "Luria Bertani" or "Luria Broth", which is incorrect. See the references 1 and 2 for more details.

For the second question: It is absolutely necessary to autoclave the media after preparation to avoid the contamination and growth of microorganisms which are present in our environment. Only making the media sterile makes sure that we can work with defined microorganisms.

References:

Studies on Lysogenesis I.
The Limitations of LB Medium


(Genetics) Is a silencer the same as gene silencing (heterochromatin)?

You are a bit confused with your terms here. Remember, when you are talking about natural regulation of a transcriptome, you can control it at the level of the genome, transcriptome and the epigenome.

The Repressor protein is a DNA- or RNA-binding protein that inhibits the expression of one or more genes by binding to the operator or associated silencers. Depending on where it binds, it regulates at the level of the genome or transcriptome.

The Gene Silencer Region, is a DNA sequence in the geneome that  that allows repressor proteins to bind to it. It is usually very near the promoter of the gene that it wants to control. This controls the expression of the gene at the level of the genome to repress the transcription of the gene. There exists a few types (Classc silencers, negative regulators and polycomb response) but lets not get into that).

An example is the regulatory element in the classical Lac operon.

Gene Silencing as you call it is epigenetic in nature. I'll point you towards the wiki as it is quite big and diverse to explain it here:
http://en.wikipedia.org/wiki/Gene_silencing

you can read more about it here:http://www.ncbi.nlm.nih.gov/books/NBK26872/


Immunoprecipitation compared to western blotting

The two techniques serve different purposes:

IP: purifies a protein

WB: visualizes or quantifies a protein

Most often when I have done IPs (in the hazy past) I have turned around and run the protein out on a WB, so one will frequently combine both techniques. Sometimes a protein is very low abundance so you will not see it in a small volume of crude cell lysate on a WB, and you have to IP it from a large number of cells to get enough protein to visualize on a WB.

Note that IPs will also co-purify associated proteins, so a good way to know whether two proteins stick together is to IP one of them and probe for the other on a WB. This "co-IP" analysis is a very common use case for both techniques.

Hope that helps.


Bacteria don't decompose the body while your alive (or even when your at the office)

Because your immune system keeps the population safe, by eating them.

Both autolysis and bacteria cause decomposition.

But there are 2 types of decomposition. The kind that keeps you from being able to be rescusitated happens within 5 minutes at room temp after death.
This is oncosis.

The kind where you smell like French cheese happens later, as the cells pop and disintegrate, and then get eaten by bacteria, which release stinky chemicals.


For question 1 it's just a biochemical response from thermoreceptor nerves in the skin, temperatures that are likely to cause lasting damage from prolonged exposure cause a neurological response - in this case it's pain.

In address to question 2 it is most likely down to the physiological response of vasoconstriction.

When the body is cold the body undergoes constriction of the blood vessels in the extremities to preserve core temperature where it is more essential for the proper functioning of vital organs. By constricting blood flow to the extremities (in this case the hands) there is less volume of blood present, because of this nerves present in the hand are closer to the surface and thus more sensitive to pain.


Why do chromosomes uncoil back to chromatin after cell divisions?

It’s happening because genetic information from DNA can be read only if it’s in the chromatin form, it can’t be read from chromosomes and so the cell can’t fulfill its functions. But if it’s need to take apart cell’s genetic material, the form of chromosomes makes it easier


Why do we exhale after we hold our breath?

This is more about basic physics than biology. When you hold your breath, you normally take in one last long breath and keep it in as long as possible, Your lungs are therefore already full of gas (remember that the oxygen used by our lungs is only ~22% of the total volume of air you inhale). Therefore, when you release that breath and want to take in a new one you need to first empty your lungs out in order to refill them.

In addition, you need to have your lungs empty in order to be able to expand them and draw in more air. There is no biological reason for any of this, it is the same principle as trying to use a turkey baster:

If you use it once and pull up some liquid into it, you will have to let air out before you can use it again. Your lungs work in the same way. Test this by exhaling all the air from your lungs and then holding your breath without inhaling. When you want to start breathing again, you'll be able to breathe directly in without needing to exhale first.


Looks like a common wolf snake, or house snake (Lycodon capucinus). Here another picture. I think they are not dangerous, but be sure to take precautions (I might have guessed wrong from just your photos).


If a hand was suddenly chopped off while holding something, would the severed hand still hold a grip?

(Disclaimer: I have no personal or medical experience of cutting off hands, or any other body parts, so this is purely theoretical.)

It seems unlikely, since most of the muscles that provide the gripping force are in the forearm and are transmitted via tendons in the wrists. Try gripping something forcefully* and notice which muscles tense up, and how the tendons on the inside of the wrist transmit the force.  Cutting the tendons would eliminate the force.

Of course anyone who's done much fencing knows that you don't grip with too much force, because that interferes with the fine control provided by the fingers.


Why should the pharyngeal cavity be essential for articulated speech?

This bit of the book Developmental Neuropsychiatry: Fundamentals describes the difference between the oral cavity in humans and other animals. It doesn't refer to Homo habilis in this context, but it says:

  As a result of this anatomical pattern, the range of sounds that an animal can make is limited because of the pharyngeal cavity, which is necessary of sound amplification, is small.

and:

  Still, this lower position of the larynx produces a larger pharyngeal space above the vocal chords, which makes possible a greater range of sound modification and becomes the key to the production of articulare speech.

Which suggests that the pharyngeal cavity has a more general use in speech than just making nasal consonants.

This article goes into some detail about Human speech anatomy and its evolution; what it says about the pharyngeal cavity is less about the cavity itself than about the position of the tongue, which allows the separation of that cavity into different sections, leading to different wavelengths being produced. They further say that apes have their tongue entirely in their oral cavity, and are as a result unable to make vowels other than schwas (the basic vowel you make when you're making no effort whatsoever) (they do mention one exception but that species apparently doesn't use its pharynx to make vowels like we do).

The article makes another very interesting point that is relevant to your question, I quote:

  Speech communication would be possible without quantal vowels [note from me: the sounds it was talking about that we can make but monkeys can't]. Indeed, there would have been no selective advantage for retaining whatever mutations led to the evolution of the human supralaryngeal vocal tract unless some form of speech had already been part of hominid culture.

That is an excellent point, which is that we need to separate the concept of "speech" from "fully modern speech". Things evolve gradually, and there will have been a continuum from simple communication and vocalizations that our latest common ancestor with other apes likely did, to modern speech with its sounds, grammar, and anatomical and cognitive abilities it implies. And since we no longer have examples of anything on that continuum, we don't know which parts of it we'd be more or less likely to think of as "speech".


Structure separating the left atrium from the ascending aorta?

There isn't any particular structure there: you have the wall of the aorta/adventitia, and if you have an explanted heart there is a space and then the auricle of the left atrium on one side and the right atrium on the other. These would all be contained within the pericardium.

Where the aorta is most "touching" the left atrium is where the pulmonary veins come in: I think this picture from Gray is most helpful.

Figure 494. Henry Gray (1825–1861).  Anatomy of the Human Body.  1918.

There really isn't much to distinguish these veins from the non-auricle part of the atrium, similar to the vena cava on the right side. If you were to cut along the veins eventually you would just open up into the atrium.

The Visible Heart Lab is another good reference http://www.vhlab.umn.edu/atlas/aorta for cardiac anatomy.


Species definition

The whole problem is based upon your definition of hybrid and therefore your definition of species. If a hybrid is the offspring of two individuals belonging to different species and if we define species based on reproductive isolation, then a hybrid can, by definition, not be fertile.

However, we often call two lineages as being two different species even if they can reproduce. To get into more difficulties we could differentiate between pre-zygotic and post-zygotic reproductive isolation assisted reproduction.

You should definitely have a look at the post How could humans have interbred with Neanderthals if we're a different species? for a discussion on the concept of species.

In practice

In practice we often call two lineages as different species even though they are not reproductively isolated. In such cases, yes hybrids are fertile. In fact, hybrid can even have a very high fitness, a phenomenon called heterosis (aka. hybrid vigour). A historically important example is one described by Darwin in The Variation of Animals and Plants under Domestication; the hybrids between the common pheasant and domestic fowl are larger nd more resistant than both parents.

Note also, Haldane's rule that states if in a species hybrid only one sex is inviable or sterile, that sex is more likely to be the heterogametic sex. This is for example the case of the Yakalo (hybrid of yak and bison).

There are also cases where the hybrids are reproductively isolated from both parent species and end up creating a third species. This is called hybrid speciation.


Yes. The chemical/hormone that causes attraction is called serotonin. That's what causes attraction between two people. Theoretically, chemicals can be used to make people fall in love with you, such as MDMA, which increases serotonin levels. If the "animal" (such as a cat or a monkey), was in the same room as you and you gave the cat MDMA and you were the only one in the room, the cat would bind with you and seratonin would be released.

This is why MDMA and methamphetamine are known to cause homosexuality and fetishism, for the drug tricks the human into thinking that serotonin is formed with a guy who he is hanging out with or what he is doing at the very moment.

Another hormone that is a "love" hormone is called oxytocin, but this chemical will only cause bonding once the love is formed.

In short, serotonin makes someone fall in love with you, while oxytocin will keep someone in love with you.


How can natural selection occur at species level whilst not occuring at the individual level?

I think you have misunderstood the passage. Here is a larger section (found at google books):

  Natural selection can also occur at the level of species, for certain characteristics enhance the rate of origin of new species or diminish the likelihood of species extinction. For instance, the number of species in lineages of herbivorous insects has generally increased faster than in closely related lineages that have other feeding habits. Neither gene selection nor species selection has molded the advantageous characteristics of individual organisms; rather, they have affected properties at the gene level or at the species level. But individual selection, selection among individual organisms within populations, is at the center of evolutionary theory. It is at this level that selection explains most of the adaptive features of organisms.

Going through this part-by-part; the first two sentences state that lineage/species selection can occur, in the sense that species traits can enhance the speciation rate of a lineage or decrease their risk of extinction, relative to other lineages (e.g. in herbivorous insects).

He is then saying that the traits that are the target of this selection are not traits of individuals but traits/properties of the species/lineage.

An example might help to explain the point. For instance, it has been argued that pelagic larvae in sessile ocean species will lead to higher dispersal rates, which means that species can colonize new environments, and this can lead to speciation through adaptive radiation (Jablonski & Hunt, 2006). A larger range will also correspond to lower extinction rates (everything else equal). The trait might also be fixed within a lineage (so there is no variance at the individual level within-species), and if so, this lineage as a whole could have higher speciation rates and lower extinction rates compared to a sister lineage that lacks pelagic larvae.

The individuals within species will naturally have the underlying traits (pelagic larvae), but the traits that are selected at the lineage level (extinction risk & speciation rate) are not properties of individuals but are traits of the lineage/species.

He then ends by continuing with "normal" individual selection, and states that processes at this level is responsible for most adaptive features of organisms. It should also be noted that lineage selection is still considered controversial, and it has been shown that it is inherently much weaker than selection at the individual level. Personally, I think there are some very good examples of how species/lineage selection can function, but to what extent it is an important process for species and organisms is an open empirical question. If you are interested to look further, Jablonski (2008) and Okasha (2007) are two good starting points.


Is there a biological mechanism for evolution encoded into our DNA?

This entire answer will be long, so read the short part first, then read the rest if you (or anyone else) is curious. Citations are included in the long section. I can include additional citations in the short section if needed.

Long Story Short

Your question touches on some common misconceptions about how the evolutionary process. Organisms don't "want" to evolve traits. Traits evolve through the biological processes of random mutation and natural selection.

Organisms do not "want" to evolve traits.  (Well, OK, I'd love to evolve an extra pair of hands but that is not possible.) Natural selection works by modifying existing traits. Your turtle can stare all she wants at food out of reach but she will not evolve a longer neck. Instead, natural variation exists among neck lengths of the turtles because of variation of the genes that determine features related to overall boxy size. Those individuals with longer necks may be able to get a bit more food, live a little longer, and reproduce a little more. They will pass along their genes to their offspring, so perhaps more of their offspring will also have longer necks. Over many generations, the turtles may have somewhat longer necks.

A common misconception is that the traits of organisms are precisely adapted for a specific need. They are not, for a few reasons. First, natural selection occurs relative to the current environment. Adaptations that work well in one environment may not be so useful in another environment. Environments are rarely stable over evolutionary time so traits are subject to constant change.

Next, as mentioned above, natural selection can only work on what traits are present. While an extra set of arms would be handy, I am a tetrapod. My four appendages, along with the appendages of all other tetrapods, trace back to our common ancestor. The appendages of all tetrapods are modifications of that ancestral trait.

Finally, organisms haven't "sampled" the entire realm of possible mutations and combinations of mutations. In other words, a certain mutation or set of mutations might actually be able to adaptively improve a particular trait in the current environment but, if the mutations never occur, then the improvement can never happen.

We only need to look at ourselves to realize how imperfectly adapted we are. We get bad backs and knees because our bodies weren't designed to walk upright. We evolved from quadrupedal organisms. This has happened so recently that changes in the structure of our knees and backs haven't yet evolved (and may never).  Search the internet for the "blind spot" eye test. We have a mass of blood vessels in front of the retina of our eyes, which reduces our visual accuity. We often have to have teeth pulled from our jaws because the flattening of our face (relative to our australopithicine ancestors) has shorted our jaws. We don't have as much room for our teeth but we have not evolved a reduced number of teeth.

As for human technology being able to make direct changes to our DNA to improve our adaptability, I would say no. While I do not have the ability to see into the future, the complexity of our genome, and more specifically how genes are regulated, suggests to me this would be a very daunting if not impossible task.  See the long answer below for more on regulatory genes but the gist is that a small set of regulatory genes control most of the other genes (including other regulatory genes). The interactions are extremely complex and we have a detailed understanding of very few of these interactions.  I speculate that affecting one such gene in a "positive" way is very likely to have many unintended negative consequences.

Below are some simple math and other ideas to show you how mutations can lead to the many adaptive traits that you see among the diversity of life on earth.

Long Story

  how can so many specific (advanced) evolutionary leaps be otherwise explained?

Mutations occur at random throughout the genome. Most mutations will be neutral. That is, they are neither bad or good from an evolutionary viewpoint. The mutations are neutral because the genome for most organisms is non-functional. Mutations that occur in the functional regions of DNA (i.e., protein-encoding and related regions) are more likely to be detrimental (bad) because the mutation may negatively affect the function of the protein or even the ability to produce the protein.  However, some mutations are beneficial. The mutation may actually enhance the functionality of the protein or even produce new proteins.

A couple of factors have to be considered regarding mutations.  The mutation rate is very low.  For example, Kumar and Subramanian (2002) compared the DNA sequences of 5669 protein-encoding genes from 326 species of mammals.  Their results suggested that the average mutation rate among mammals is 2.2 x 10$^{-9}$ per base pair (bp) per year.  This means that, on average, a point mutation has changed each DNA nucleotide position in the mammalian genome slightly more than twice (2.2 times) every billion (10$^9$) years. That's a lot of time!

However, this same rate occurs in every individual in the population, so you have to consider the population sizes of the organisms. So, let's do a simple exercise. Consider a species like the rock pocket mouse or another small mammal that has a very short generation time. For this simple example, let's assume the generation time is one year. That means that the mutation rate of 2.2 x 10$^{-9}$ per bp per year would then correspond to 2.2 x 10$^{-9}$ mutations per bp per generation. Generation time is important because new mutations are inherited only through reproduction.

Assume the average mammalian diploid genome is about 6 billion (6 x 10$^9$) nucleotides in size. The number of heritable mutations that occur in a single offspring is

$$(6 \times 10^9) \times (2.2 \times 10^{-9}) = 13.2.$$

Next, assume that about 2.5% of the mammalian genome is composed of functional, transcribed sequences that may affect the phenotype (the traits of the organism). That means that, of all the mutations that occur in every offspring every generation, about 2.5% could potentially affect the phenotype.  That is,

$$13.2 \times 0.025 = 0.33.$$

Still a small number.  But, now we have to account for population size. Small mammals, like mice and voles, generally have large population sizes.  Assume that the population of rock pocket mice contains 100,000 reproducing individuals. If so, then

$$0.33 \times 100,000 = 33,000,$$

which is the number of new heritable mutations that could occur in the population. Most of these mutations will be detrimental and removed from the population by natural selection but, if even a small fraction of these new mutations are beneficial, then natural selection can cause these beneficial mutations to increase rapidly in frequency in the population during future generations.

In humans, Nachman and Crowell (2000) estimated that the average mutation rate was 2.5 x 10$^{-8}$ mutations per bp per generation (not year), by comparing the genomes of humans and chimps. If we assume the same genome size and effective human population size of 500,000 individuals, then applying the same math suggests that 1,875,000 new mutations that potentially affect phenotype occur in the human population every generation. Again, only some of these will be beneficial but that is still the possibility of a number of new beneficial mutations. In evolutionary terms, a mouse or human generation is the blink of an eye.

How long would it take for a beneficial mutation to spread through a population? That depends on two things. How beneficial is the mutation (called the strength of selection, s) and the population size? To estimate how long it would take for a beneficial mutation to spread through a population, we can use the formula,

$$t = \frac{2}{s}\mathrm{ln}(2N_e),$$

where $t$ is time in generations, $s$ is the strength of selection, and $N_e$ is the effective population size (number of reproducing individuals). For the strength of selection, let's assume $s=0.01$, which is weak but positive natural selection. Going back to our rock pocket mice with $N_e = 100,000$, then the beneficial mutation would be spread throughout the population in only 2441 generations (remember, we're talking evolutionary time so 2000 years is nothing). If $N_e = 10,000$, the mutation spreads in only 1981 generations.  If we increase the strength of selection t 0.2, then the times are 122 and 99 years for population sizes of 100,000 and 10,000 years, respectively.

These "back of the napkin" calculations show just how quickly even weakly beneficial mutations can appear and spread throughout a population. Yet, this doesn't include other types of mutations like gene duplications that can also allow new proteins to evolve. For example, human ability to see red colors is due to a simple gene duplication (Nathans et al. 1996 and references therein). This duplication also explains the common form of red-green colorblindness.

Whew!

There's yet more to our mutational story. Consider humans and chimps, which are nearly identical from a genetic standpoint (between 96-99% depending on how you calculate it) yet they appear very different.  If humans and chimps diverged from their common ancestor within the past five million years, how could they differ so much? This question was initially posted by [King and Wilson (1975)]. They argued that mutations to structural proteins (like those that compose bones and muscles) would not be enough to explain the phenotype differences between humans and chimps. The proposed that regulatory genes are the key to understanding the big differences. Regulatory genes are those that control other genes, by turning them on or off and other important functions. Changes to the regulatory genes can cause fairly rapid changes to the phenotype.

This understanding has led to the broad (and fascinating) field of evolutionary developmental biology. This field focuses on how mutations in regulatory genes associated with development (from embryo to adult) have had a long-term evolutionary impact. The field is rich with examples, but one cool one is associated with duck feet and bat wings. Let's begin with the embryo. Most vertebrate embryos have membranes between the digits (fingers and toes) during an early stage of development. For most vertebrates, the membranes are lost later in development. The small flaps of skin you have between your fingers are the remnants of your embryonic membranes.

A set of regulatory genes called BMPs (and a couple of others) are responsible for causing the loss of the membrane in vertebrates. However, through different sets of mutations, the BMPs are not able to function in duck feet and bat hands. Thus, they both end up with membranes between their digits (Weatherbee et al. 2006).  Thus, two different mutations block the same set of developmental genes, leading to novel adaptations in two very different types of vertebrates. One final example is the evolution of bird feathers from scales. As you may know, birds are evolved from dinosaurs. It turns out that bird feathers and alligator scales (alligators are birds closest living relative) use the same regulatory genes to develop. The genes are BMP2 and SHH (sonic hedgehog for fans of the old computer game) (Harris et al. 2002). Other regulatory genes underlie the different types of feathers, like downy feathers and flight feathers (Harris et al. 2002).

Literature Cited

Harris, M.P. et al. 2002. Shh-Bmp2 Signaling module and the evolutionary origin and diversification of feathers. Journal of Experimental Biology 294: 160-178.

King, M.-C. and A.C. Wilson. 1975. Evolution at two levels in humans and chimpanzees. Science 188: 107-116.

Kumar, S. and S. Subramanian. 2002. Mutation rates in mammalian genomes. Proceedings of the National Academy of Sciences USA 99: 803-808.

Nachman, M.W. and S.L. Crowell. 2000. Estimate fo the mutation rate per nucleotide in humans. Genetics 156: 297-304.

Weatherbee, S.D. et al. 2006. Interdigital webbing retention in bat wings illustrates genetic changes underlying amniote limb diversification. Proceedings of the National Academy of Sciences USA 103: 15103-15107,


After more reading and information its quite simple.
our eyes picks up the energy from the primary colors.
if a television has higher BLUE energy than RED and GREEN that picture will have a blueish cold look to it even if its calibrated to D65

the SPD from television nr1 has higher energy for GREEN and RED and so whites on this television will look more warm and less blue.

how distinct and separate the cones are can also matter here.
television nr1 has no clear separation between GREEN and RED while television nr1 has.


Why there is a very high drop in pressure from arteries to arterioles?

The vessel diameter is the most principal determinant to control resistance. Compared to other smaller vessels in the body, the artery has a much bigger diameter (4 mm), therefore the resistance is low.

In addition, flow rate (Q) is also the product of the cross-sectional area of the vessel and the average velocity (Q=AV). Flow rate is directly proportional to the pressure drop in a tube or in this case a vessels, although there are other factors of blood vessels that contribute towards the difference in pressure drop in bifurcations of blood vessels. These include viscosity, length of the vessel, and radius of the vessel.

Factors that determine the flow's resistance as described by Poiseuille’s relationship: ∆P=8µlQ/πr4:

∆P: Pressure drop/gradient µ: Viscosity l: length of tube. In the case of vessels with infinitely long lengths, l is replaced with diameter of the vessel. Q: flow rate of the blood in the vessel r: radius of the vessel

Assuming steady, laminar flow in the vessel, the blood vessels behavior is similar to that of a pipe. For instance if p1 and p2 are pressures are at the ends of the tube, the pressure drop/gradient is (p1−p2)/l =∆P.

In the arterioles blood pressure is lower than in the major arteries. This is because velocity of flow is increased with decrease in diameter and vice versa.

Source: http://en.m.wikipedia.org/wiki/Blood_pressure_drop_across_major_arteries_to_capillaries

This is important as for efficient exchange at the capillary bed pressure and flow need to be much lower.


Webtool to design guide RNA (gRNA) for use with CRISPR-AsCpf1?

Most online tools will only help you to design gRNAs for Cas9 because it is the one that is most commonly used. I found an online suite called RGEN tools that also has an option for AsCpf1.

However, if you don't find it satisfactory then my suggestion is that you either design your gRNA manually or write your own script (which won't be that complex).


What species of spider is this? Could anyone help

This is a male Carrhotus sannio (the females are brown/grey). The white dorsal stripe on the cephalothorax is right beneath the outside eye; the upside-down v-marks on the back of the abdomen point to two white lines (sometimes broken) which run parallel to the length.
Gallery of males:
https://inaturalist.ca/taxa/334660-Carrhotus-sannio/browse_photos?quality_grade=any&term_id=9&term_value_id=11


What does it mean for muscles origins and insertions to be the other way around in the right than left limb?

Muscles span joints, and their purpose is to affect movement of that joint. Typically a muscle's origin is the more fixed or stable point of attachment to bone, while the insertion is on the bone which moves when contraction or relaxation of that muscle occurs.

Usually with the muscles of the limbs we think of the more proximal end of the muscle as being the origin, and the distal end being the insertion, because muscle action tends to move bones more distal to the limb joints. For example, for movement of the elbow joint you can typically think of muscles originating on the humerus and inserting on the radius and ulna.

However the nomenclature is somewhat arbitrary, because one can in a sense reverse the origin and insertion by fixing the location of the more distal bone and moving the upper body. For example, by placing your forearm still on the table and moving your upper body, because of the nature of their action in this position you might consider the muscles of the elbow to originate on the radius/ulna and have their insertion on the humerus.

I suspect this is what is described in the book. The biceps brachii attaches to the scapula and radius. Depending on whether it is the forearm or the shoulder moving with respect to the elbow joint, the insertion and origin could be considered at either end of the muscle. It is not that the origin/insertion is always opposite for the right versus left limb.


At what cancer stage do tumors release circulating tumor cells into the blood?

Tumour cells can also metastasise using the lymphatic system. They first need to detach (produce enzymes that break down the extracellular matrix or any adherent molecules etc). Then they need to survive in the blood which is really hard with all the mechanical stress of being pushed around, the completely different nutrients, the different levels of blood gases, the immune cells in the blood and numerous filter mechanisms, then to truly metastasise they need to settle down somewhere and grow in their new environment. That's a lot of things right? So a sensitive assay like one you describe would need to have that tumour cell in the sample (unlikely, and only one cell is required to make a new tumour), is likely to be unable to tell that the cell is a tumour cell, wouldn't be able to differentiate between tumour cells that could or couldn't survive in the blood or differentiate those that could then go on to grow more in a tissue or not. And of course you're missing the entire lymphatic system. Following all of that, why does it matter? See a cancer, cut it out if you can and give chemotherapy to get rid of any possible cells outside of what you cut. If your assay came back negative or positive, treatment wouldn't change.


How hard is it to build a promotor in a DNA that initiates DNA transcription when specific hormones are around

Promoters bind transcription factors (TFs) and RNA polymerase (or a related complex, in the case of eukaryotes).

The substances you are interested in testing would need to bind to DNA in the same way as TFs and RNA polymerase, in order to trigger RNA synthesis (which you might measure with RNA-seq experiments). That would seem to severely limit what you can test the presence of.

In any case, if you could make that happen for your substances of interest, you would probably also want to make sure to avoid degradation effects of short-length RNA, by removing associated proteins.

https://en.m.wikipedia.org/wiki/Promoter_(genetics)

https://en.m.wikipedia.org/wiki/RNA-Seq

https://en.m.wikipedia.org/wiki/Small_RNA


These chemo-synthetic bacteria gets energy from oxidation of inorganic compounds. Following table from Biology of Prokaryotes shows main reactions used by different species,

This oxidation happens only if electron acceptor is available. These all reactions have negative Gibbs Free Energy, hence they are favorable reactions and happens spontaneously. Generally oxygen is preferred electron acceptor but in case of lack of oxygen nitrate, sulfate, $HCO^{3-}$ etc are used.


Confusion about resting membrane potential and the Na/K pump

The Sodium/Potassium pump maintains the overall concentrations of ions on either side of the membrane. In other words it keeps the neuron's battery charged.

Imagine the neuron like a circuit. We can imagine that the electro-chemical gradient for each ion species (Potassium, Sodium, and so on) as batteries. So When the ions flow according to their electrochemical gradient, the potassium current flows outward and sodium  current flows inward as dictated by these batteries reversal potentials [negative for potassium, positive for sodium].

However, note that this will discharge those batteries, i.e. the concentration of sodium on the outside of the cell will drop (slightly) as sodium flows inward the cell, and potassium flows outward the concentration on the inside will drop slightly. Thus the magnitude of the reversal potential of the Sodium and Potassium drops (they move closer to zero).

To counteract this discharge the Sodium/Potassium pumps expends energy in the form of ATP in order to recharge the batteries. The amount of current that the pumps moves is very small relative to the larger currents of sodium rushing in and the potassium rushing out during an Action Potential. Thus when a neuron fires there is very little overall current contribution from the pumps.

When the neuron is at rest (not firing) then the Sodium/Potassium pump is able to recharge the membrane and the current becomes important. Still this is a very slow process, relative to action potentials.

Another thing to note, even though the cell is at rest the batteries are discharging slightly. Their is a leakiness to the membrane, sodium leaks in and potassium leaks out. Thus the pump's current will counteract these leaky currents at rest, keeping the batteries charged and thus the overall net current  across the membrane at rest will be zero.

Remember though, these currents are very small currents.

EDIT: Based on Discussion with @Christiaan, I decided to post a update to clarify.

At rest potential Sodium Ion influx is much smaller than potassium ion efflux and thus can be ignored. Whether or not Sodium is ignored the above still holds true. The neuron's membrane is at rest which means the net current is equal to zero. This means that the outward potassium current is counterbalanced by the pump's current thus keeping the relative concentrations of Potassium and Sodium constant while at rest and maintaining a steady rest potential.

EDIT 2: Clarifying as per OP comment:

So during an action potential Sodium and Potassium ions move in large currents and this current flow discharges the batteries. At rest there is still current flowing (thus smaller discharging of the battery) but there is no Net current flow thus no change in voltage(thus the neuron is at rest). It is exactly counterbalanced by the sodium/potassium pumps. The reasons the pump doesn't overcharge the batteries is the constant but smaller discharge at rest. When the recharging current is equal and opposite to the discharge via the leak current, a equilibrium is reached.

However when lots of action potentials fire, the resting potential will be altered from the larger discharge rate, thus when the neuron returns to its new resting potential the leak current and pump current will not be balanced, thus the recharging pump current will be larger in magnitude and will bring resting potential back to its normal value.  (Note what constitutes large is relative between species and even neurons.)

Edit 3: Rates of Leakiness and Pump activity.

If only you could say one is stronger than the other. It depends on the conditions of the cell.

Lets focus in on potassium. The leak current for potassium is dependent on two (really three) things. The permeability (or conductance) to the membrane to potassium, and the reversal potential of potassium (which is dependant on the internal and external Concentration of Potassium. Thus varying these things will strengthen/weaken the power of the leak current.

The Sodium Potassium Pump is dependent on three things. The External Concentration of Potassium, The internal concentration of Sodium and the availability of energy (in the form of ATP).

The point where these two strengths are about the same is when there is more Potassium on the inside of the cell. That is to say pump is capable of keeping up with the leakiness of the membrane until the internal concentration of potassium reaches about $140$ $mM$ (again that just a ballpark number). If their is less Potassium in the cell then $140$ $mM$ the pump is stronger, if there is more than $140$ $mM$ the leak of potassium is stronger.


How is evolution possible in contemporary humans?

It is certainly not true that "all male humans have roughly 2.5 (or whatever the number) kids". First of all, male and female humans have exactly the same reproductive rate. For obvious reasons, every time a male has offspring, a female must have had also. Last I checked neither male nor female humans are capable of parthenogenesis (certain popular religious beliefs notwithstanding).

Second, let's assume that the 2.5 number is correct. That would be the average number of children per couple. That does not mean that all couples will have 2.5, or even that most couples will have 2.5. It just means that the average will be 2.5. If, for example you have one couple with 6 children, one with 2 and two with 1, the average will be (6+2+1+1)/(1+1+2)= 2.5.

On to the main point. What does selection mean? In its simplest form, that the individual most likely to survive (the famous "fittest") is also most likely to reproduce. This is a very simple concept, the longer you live the higher your chances of managing to have offspring. If you die two weeks after birth it is going to be hard to manage to reproduce yourself. This has not changed.

So, what does "fitness" mean? It can mean many things. If you are a warm blooded creature at the beginning of an ice age for example, it could mean being better at regulating your temperature than your peers. If you are a 21st century human, it could mean being funnier on twitter than your peers. The two are not fundamentally different. They can both be selected for or against. As long as one mate is chosen over another, selection is happening and the "fittest" (in each particular context) is most often selected.

  Add in the process of culturally modified selection pressure, and it
  seems to me that even an "unfit" male would end up having a couple of
  offspring. The fittest male (or female) is no better off than his or
  her contemporaries because of this "leveling" effect.

"Culturally modified selection pressure", as you call it, is still selection pressure. Cultural factors can change what it means to be "the fittest" but there is no objective gold standard of "fitness". While it may be true that in modern human society, different characteristics are selected for than was the case with early Homo sapiens, this does not mean that "evolution is not occurring". On the contrary, it is occurring but perhaps it is moving in a new direction. In fact, this is essentially a circular argument. By definition, "fittest" means most likely to survive and reproduce. It does not mean strongest or fastest or prettiest. It just means whoever is better at reproducing. If that happens to be those individuals who are best at square dancing, then it is they who are the fittest.

Take the example of a modern human with diabetes. Medicine allows diabetics to lead fully productive and largely normal lives. So, perhaps diabetes is no longer a selective criterion. This does not mean that the diabetic cannot be selected for or against based on their fitness on other scales.

Whatever the selective pressure, whatever it may be that defines a "good mate", if selection is present then so is evolution. The only way to remove a species from the process of selection would be to have all (or none) individuals of each and every generation reproducing at the same rate. This is clearly not the case with humans. Surely not everyone around you has, or will have, children? There you go, selection!

UPDATE:

In answer to your comment, yes indeed, in order for a selective pressure to make itself felt and affect phenotype (at the species level), it needs to be constant across several generations. However, even the absence of selective pressure affects evolution. As others have mentioned below, active selection is not the only mechanism of evolution.

Your main question however seems to be the following: If modern society (medicine etc) allows individuals that would not survive in the wild to reproduce, how does that affect evolution? The main points in my answer, and all others here, are:

Even if we accept that modern humans have removed themselves from the purely "biological fitness"-based selection pressure (an assumption I am not at all sure is true), and assuming that this removal is constant enough over many generations (again unclear), even if all this is true, evolution is most certainly still occurring. It may even be faster since genotypes that would not survive in the wild persist in the gene pool, thereby increasing its diversity.
As you point out in your comment below, for such social pressure to make itself felt, it needs to be constant across many generations. We are probably not there yet.
Most importantly, as I said above, there is no such thing as an absolute biological fitness. When the ecosystem changes, so does the definition of fitness. Modern humanity's ecosystem, our habitat, is intimately connected with our culture and society. If an individual is better at reproducing in that context, then that individual is more fit.


What percentage of twins has the same gender

You stated:

30% of twins are identical
50% of non-identical twins have the same gender

Therefore 70% of twins are non-identical and 50% of those will have the same gender. I'll leave the rest of the calculation to you.


How does vasoconstriction/vasodilation change blood pressure?

The circulatory system is a dynamic system which cannot be adequately explained by your example (at least not by me). You need to understand it, not seek to make it fit your (especially) electrical circuits with resistors. Blood isn't electricity. At least try a fluid dynamics model.

Let's take this very simple model: Blow up a balloon four-fifths of the way, and put a wide inflatable cuff around its middle. Inflate the cuff so that the balloon bulges just a tiny bit at the ends, then stuff it all into a plexiglass box so that the balloon has no room to expand. The plexiglass box represents our body. Lets call the pressure inside the balloon now normal blood pressure. The air in the balloon represents your total blood volume. It can't change from moment to moment; it's fixed. The box can't change it's volume moment to moment either. It's fixed.

The cuff represents arterial smooth muscle. Vasoconstriction can be represented by inflating the cuff further. Constriction (inflation of the cuff) will increase the pressure throughout inside the balloon, because the same amount of gas now has to exist in a smaller space. Vasodilation (deflating the cuff) decreases the pressure inside the balloon, because the gas can now inhabit a larger amount of space.

That's it, really. If the same volume must inhabit a smaller, constricted space, the pressure exerted by blood in that space will be higher. If the blood vessles dilate, the pressure in the blood vessels falls.

Now add about 20 layers of complexity to that simple model, and you have a working model of the circulatory system.

Sparknote answers:

Arterial and arteriolar smooth muscle "injects energy into the system", resulting in systemic increases in the pressure existing in the vascular "circuit" (meaning somewhat elastic tubing) if resistance requires energy. (So I have misunderstood: see @Raoul's answer.)
Sorry, I didn't read this properly the first time around. Yes, the heart supplies the energy. The contribution of the elastic walls of the arteries is not active, but passive.
Absolutely. The more elastic/compliant the arteries are, the less work the heart must exert to pump the blood through the circuits. The stiffer and narrower the arteries are, the harder the heart must work to pump blood through the circuits. The result of that increased work is a thickening of the muscular walls of the heart, called ventricular hypertrophy, which is a sign of elevated pressure in the system.
I'm not sure I want to commit here to your lingo, but the answer should be inferable from numbers 1-3. Elastic vessels help (by rebound compression) to propel the blood through the circuits. Stiff vessels hinder it, making the heart do more of the work.

I am not an engineer, so I may be misusing some of the terms.

Edited to add: Please see @Raoul's answer for a better explanation.


Why do humans have digestive system present at front and kidneys back?

I don't claim to have the full answer to why these organs are located in the way they are, but I think these factors have an influence on the anatomy:

1- The urinary system's link with the reproductive system in male mammals: The male reproductive system outlet needs to be located as far to the front as possible in a 4 legged mammal to facilitate sexual function, forcing the urinary system to follow

2- The kidneys are very fragile and need to be protected by the thick back muscles and partly under the rib cage. They would be too vulnerable at the front

3- The urinary system is generally sterile of bacteria, and so is the reproductive system in males, and partially in females. The digestive system isn't sterile in both. The outlet of the digestive system can't be in the front of the genitals in a male as this will increase the risk of urinary infections overall and also genital infections after mating. It can't be in between the urinary and reproductive outlets for the same reason, so it has to be located behind both to cause the least interference with their functions


This is probably Araneus marmoreus var. pyramidatus.

Here is a better image for comparison:

Source: http://www.uksafari.com/marbled_orb_weavers.htm

PS: Despite A. marmoreus being found in US, this variety (pyramidatus) is  normally an European one. I don't know any observation of it in US. However, I'll leave this answer here until another ID is provided.


Estimation of number of species at an earlier time

according to the National Science Foundation press release"Species Have Come and Gone at Different Rates than Previously Believed" indeed show what Atl LED suggests, that the progression is not linear, with the study showing that:

  The previously thought exponential increase in diversity is not there.

Further, there is the "Palaeobiology Database, that

  seeks to provide researchers and the public with information about the entire fossil record.

The site also has links to further papers of the topic.


Why would it be beneficial for cortisol and corticosterone to exhibit pronounced anti-inflammatory activity?

Interesting question. It's easy to imagine why you would want to suppress inflammation in a fight-or-flight situation. Inflammation can cause painful swelling, fever, and it can dull mental acuity, all of which might hurt your chances of a win or escape.

After a little PubMed search, it looks like sympathetic adrenal responses (like cortisol) can actually induce a net hypercoagulability of the blood (1). So, even though inflammation does induce coagulation, it looks like the sympathetic anti-inflammatory response is not necessarily synonymous with anti-coagulation. This also makes sense in the context of Cushing's Syndrome, where hypercoagulation is associate with elevate cortisol levels, among other factors (2).


Cell differentiation by non-identical copies

I found a partial answer to my and this question at Quora:

What regulates initial cell differentiation?

Ian Driver says:

  In many organisms the initial cell has mRNA (or protein) partitioned
  in one half of the zygote.  One daughter cell inherits most of the
  mRNA or protein and goes on to activate or repress genes in that cell,
  but not in the other.  This has been visualized and well studied in C.
  elegans: Asymmetric cell division and axis formation in the
  embryo.


Is the technology "identitas v1 forensics chip" related to microarray technology?

Interesting question. The answer lies encoded in our DNA. If you take eye color for example, the genes important here are well known. The most important are OCA2, HERC2, SLC24A4 and TYR.

These are involved in different parts of the pigmentation process, any mutations in these genes lead to changes in pigmentation. Since also the variants, which lead to a altered pigmentation are known, you only need to analyze some cells containing genomic DNA from your suspect to check for these mutations (or SNP, single nucleotide polymorphisms).

The same is true for hair and skin color. So if you identify one or several mutations in the MC1R gene, you can be pretty sure, that your suspect is red haired, has pretty light skin and a high chance for green eyes.

This technique doesn't need to know the exact expression of a certain gene, but needs information on the cause for a phenotype (like skin, hair or eye color) and the causative gene.

The "Identitas v1 forensics chip" is a microarray chip, unfortunately, the company website is not really a source for information. You can find some in the New Scientist article you linked. This is basically a microarray (or chip) where all the forensically interesting and known SNPs about how a person looks like are analyzed.
It gives basic information (or at least a chance) how a persons looks like, what CSI does in the series is usually widely exagerated.


Absolutely, this is actually looking more as an engineering problem rather than a biology one.
In any case, I think I have found a satisfactory answer on a paper from the 1997 Proceedings of the Sixth European Symposium on Space Environmental Control Systems. The article is called: Oxygen Scrubbing and Sensing in Plant Growth Chambers using Solid Oxide Electrolyzers.
Basically, a disk of non-porous yttria stabilised zirconia (YSZ) is sandwiched between platinum electrodes and inserted in the chamber half-way, with the other half enclosed in whatever will store the extracted oxygen. The electrodes must be porous to allow gas diffusion. The only problem is, that sort of electrolysis cell needs to be maintained at an elevated temperature (800 to 1000 Celsius) in order to work.
By means of electrocatalysis and thermal dissociation the oxygen molecule dissociates to form two oxygen atoms, these in turn pick up two electrons from the cathode and become ions. The ion is then transported by the electrolyte (YSZ) by means of electron vacancies located in the crystal lattice (basically a similar process to that of a N-doped semiconductor whereby impurities create electron holes).
I think I am satisfied with the above explanation. Doable at home? No way, but it does answer the core question.


How do plants 'tell time' for circadian rhythms based on a ~24 cycle?

The short answer is that it is because the reaction chains dispensing time and enzymes doing those reactions have evolved to negate the impact of environmental factors.

The true answer is that the plant clocks do gradually lose sync with 24h cycle when put in stable lighting conditions and move to some more-less random characteristic frequency of their  molecular clock which is quite likely dependent on environment (still it is obviously close to 24h) -- the sad truth is that every clock needs syncing to work properly.


Simple Mendelian sex-linked chromosome fruit fly question

I think the text is slightly misleading. But before going into the detail genetics of eye color let's answer your first question.

First, wouldn't recombination scramble the eye color alleles among the sex chromosomes?

Two X chromosomes do recombine but one X and one Y chromosome do not recombine (to the exception of the so-called pseudo'autosomal region).

I am not sure why recombination would change anything here. I think the eye color of flies in determined by a single locus.

How is this possible?

Let's go through the text slowly because I think the text is a little misleading. I am suggesting the only scenario I could think of that would explain the observed pattern. Note that this scenario also depends on the interpretation of the text and I would need more info on the experience to make sure my interpretation is right.

  When he mated red-eyed flies (dominant) with white-eyed flies (recessive), most, but not all, of the F1 progeny were red eyed.

Actually, only males are sometimes white eyed here. And to be more accurate, if sex was uncorrelated form eye color in the parental lines, then half of the males should have white-eyed.

What was happening here is that red is dominant, white is recessive and the locus for eye color exists only on the X chromosome. As a consequence, all F1 females are now heterozygous (and display the dominant red-eyed phenotype) and all males are hemizygous, half of them having the red-eyed phenotype while the other half have the white-eyed phenotype.

  Furthermore, when Morgan mated the red-eyed males of the F1 generation with their red-eyed sisters, [..]

Let's forget about the white-eyed males from above, we will just cross the red-eyed males with their sisters.

  they produced about one-quarter white-eyed males, but no white-eyed females.

By they produced about one-quarter white-eyed males what is meant is that a quarter of all individuals were white-eyed males. That is half of the males were white-eyed. The other three quarters of the individuals were red-eyed.

The males received their X chromosome from their mother only (as they can only receive the Y chromosome from their father). Half of the males have therefore received the white-eyed allele while the other half have recevied the red-eyed allele. For the females, they all received from their mother either a white-eyed allele from their mother or a red-eyed allele. As we selected only father that had red eyes in the previous step, all father carry exclusively the red-eyed allele. As a consequence, all females receive a red-eyed allele from their father. Therefore half the females are homozygous red-eyed or heterozygous. In any case, all females are red-eyed.


Is it possible to identify this chrysalis?

Based on the image and location, I'd say you've got the chrysalis of a bagworm moth (family Psychidae) or some related species of case-building moth.

There are over 1350 species of Psychidae, and I am not certain of the exact species.

Caterpillars of this family are known to create protective cases out of local debris. Because this debris can vary, so too will the appearance of the case. Different species likewise prefer to use different materials. Sticks, leaves, tree scales, etc. have all been known to be used in their chrysalii. As a result, Googling for images should produce a wide variety of chrysalis structures. Here is one example using Eastern Redcedar scales in NC:

The University of Arkasnsas has a locally-relevant page about Thyridopteryx ephemeraeformis, one species that is often considered a "pest".


Gender and age-specific mutation rate in plants

From Whittle and Johnston (2006):

  Specifically, human epidemiological data and/or nucleotide substitution rates of selectively neutral DNA (which equals the mutation rate, Kimura, 1983; Miyata et al., 1987) have shown that more mutations occur in the male than in the female germ line for numerous animal taxa (e.g. humans, mice, chickens, and sheep) and in older rather than younger human males, patterns that each agree with the cell-division hypothesis (i.e. more DNA replications in males and in particular older males; Penrose, 1955; Risch et al., 1987; Becker et al., 1996; Moloney et al., 1996; Li, 1997; Green et al., 1999; Crow, 2000; Li et al., 2002; Makova and Li, 2002). Other data, however, have indicated that the mutation bias reported relative to gender and male age are not generally well correlated with the number of germ cell divisions and that other factors could explain these trends, such as methylation patterns, differential repair, metabolic rates, and preferential transmission of mutations to pro- geny from older males (Risch et al., 1987; Martin and Palumbi, 1993; Drost and Lee, 1995; Bromham et al., 1996; Hurst and Ellegren, 1998; Martin, 1999; Crow, 2000; Huttley et al., 2000; McVean, 2000; Sommer et al., 2001;Hebert et al., 2002; Hurst and Ellegren, 2002; Kumar and Subramanian, 2002; Li et al., 2002; Bartosch-Harlid et al., 2003).

…so basically we don't really know whether difference in gametogenesis between males and females lead to more sex and age-specific mutation rate!

However, Whittle and Johnston (2006) in reviewing many articles also show that old seeds are more likely to carry chromosomal aberration and that old individuals are more likely because of the instability of their metabolism to transmit more mutations but that would be sex independent and would not result in different gametogenesis as I was expecting in asking this question. Also the kind of mutations that become more frequent with all parents are chromosomal abnormalities or important deletion but not so much substitutions (which was also what I was interested in when asking).

Thanks @DevashishDas for finding this article. This other one was interesting as well.


If mammals evolved to be nocturnal, then how did we get so staunchly diurnal in such short time?

The Nocturnal bottleneck for mammals as it's called was assumed 'til recently to have only ended at the Cretaceous–Paleogene extinction event 66 million years ago.

At the basis of the nocturnal bottleneck hypothesis lies the idea that
Mesozoic ectothermic reptiles (including dinosaurs) would be
restricted to daytime activity at times when the sun can help to heat
up their body to operating temperatures.......

The arguments against:

include:

(i) presence of insulating proto-feathers,

(ii) fossils of breeding dinosaurs in cold regions

(iii) raised body posture

(iv) fibrolamellar bone structures

(v) presence of nasal turbulate bone structures

(vi) stable growth rate (isotope deposits in bones and teeth)

(vii) possible ‘mass homeothermia’ or ‘inert homeothermia’ in large dinosaurs

(viii) the occurrence of small dinosaurs in cold regions

(ix) the existence of potential external thermoregulatory structures in some dinosaur species (e.g. dorsal boney plates in Stegosauria).

Recent molecular analyses have added to the doubt of the original theory:

saurischians are now generally believed to have had partial
endothermia, allowing them to incubate their eggs and inhabit colder
regions......
Independently, parallel evolution in Synapsida led to partial
endothermia in therapsid reptiles (250 Ma, Late Permian) and full
endothermia in mammals

Nespolo RF, Bacigalupe LD, Figueroa CC, Koteja P, Opazo JC. 2011. Using new tools to solve an old problem: the evolution of endothermy in vertebrates. Trends Ecol. Evol. 26, 414–423
The implication being that the pure nocturnal adaptation in mammals would not be a completley effective strategy for predator evasion.

In a large comparative study [Roll], divided 700 species into classes of
dominant temporal activity niche: nocturnal, diurnal or both (mostly
crepuscular). They used an existing phylogenetic construction to
reconstruct the evolution of diurnality in rodents, which are thought
to have diversified after the K/T boundary. The reconstruction of
temporal niche usage within rodents led to the conclusion that rodents
shared a nocturnal ancestor that existed before the K/T boundary.
Subsequently, diurnality evolved through secondary evolution at least
seven times independently in Rodentia.
Using the molecular phylogenetic construction of [Huchon et al],
we constructed the temporal niche phylogeny within the full therian
subclass using the nocturnal, diurnal and
crepuscular/arrhythmic classification of [Roll et al.]. The most
parsimonious interpretation leads to a minimum of 16 changes of
dominant temporal niche.

Bolding mine.

Most subdivisions of Theria are indeed nocturnal, but many changes and reversions occurred, and different interpretations may be
possible, especially when higher-quality data on activity patterns in
more species become available.

Conclusion.
It apears that mamals may have been chased by the saurischians throughout the day, night and twilight hours and that many adaptations and reversions may have occured over a period (possibly) much longer than the 66 million years previously thought. Research is ongoing.
Bolded references:
Roll U, Dayan T, Kronfeld-Schor N. 2006. On the role of phylogeny in determining activity patterns of rodents. Evol. Ecol. 20, 479–490
Huchon D, Madsen O, Sibbald MJ, Ament K, Stanhope MJ, Catzeflis F, de Jong WW, Douzery EJ. 2002. Rodent phylogeny and a timescale for the evolution of Glires: evidence from an extensive taxon sampling using three nuclear genes. Mol. Biol. Evol. 19, 1053–1065


Could all mammalian receptors be described as allosteric?

"Allosteric" refers to an alternate site of action, but it is not limited to a discussion of enzymes. For a protein that is a receptor, allosteric modulation refers to modulation of that receptor away from the binding site or "active site" of the endogenous ligand.

Although it is true that the receptor may have enzymatic activity when a ligand is bound, it would be non-standard to use the term "allosteric" to refer to the normal ligand binding site, even if this terminology is technically correct with respect to the enzymatic activity.

There are many receptors which are not enzymes at all: a great example would be ligand-gated ion channels - all of the activity of these proteins is due to their conformational changes, and not due to any catalyzed reaction.


Drosophila is seen as a highly alcohol tolerant species which is mainly dependent on the environment it lives in. So are flies, which are captured in the cellar of a wine yard more tolerant to alcohol than flies which are captured outside (see first reference).

The environment in which the flies grow up and live does not influence the activity of the alcohol dehydrogenase enzyme but seems to influence the expression (amount) of it. The more alcohol is present, the higher the expression is (see reference 2).

I haven't seen explicit data on the comparison between humans/mammals and drosophila, but since drosophila is seen as a good model organism to study alcoholism, I would think it is not too different. (see reference 3).

Micro-differentiation in a Natural Population of DROSOPHILA
MELANOGASTER to Alcohol in the Environment
Alcohol tolerance, ADH activity, and ecological niche of Drosophila
species
Functional Ethanol Tolerance in Drosophila


Does the deletion of Chromosome 20 cause immunity against prion disease?

I see one report of a viable autosomal monosomy,

(https://www.nejm.org/doi/full/10.1056/NEJM196710122771502)

but in general, they are not survivable.

https://www.ncbi.nlm.nih.gov/pubmed/4086989
https://www.biology.iupui.edu/biocourses/N100/2k2humancsomaldisorders.html
https://www.ncbi.nlm.nih.gov/pubmed/29164644

A better question would be if person with a deletion or natural knockout of that gene are susceptible to that prion disease.  Those subjects likely exist in larger numbers, though it's likely that few have been exposed to any particular prion disease.


You are being too smart for this question ;) If you assume that the enzyme is perfect, you've optimized the amount of template, and you leave the reaction for a really long time then there will be complete digestion. If you assume complete digestion, then there won't be any 1.1kb fragments. Furthermore, if you work out all the possible orientations of the 800bp and 300bp that were ligated together (each piece can be in the orientation shown and the opposite orientation) and you force an EcoRI cut at every available EcoRI site, then you won't have any 0.4 kb fragments from imcomplete digestion, either.

In reality, I've never been able to optimize my reaction conditions to get complete digestion of my template, and I always have some residual template around, so it's good your thinking that way for "real-world" preparation.

If you do not assume incomplete digestion, in addition to the 1.1kb and 0.4kb bands missing from this gel, I calculate that there should be a 1kb band as well.

Edit: I goofed up the last paragraph. It should read: "If you do not assume complete digestion, in addition to the 1.1kb and 0.4kb bands missing from the complete digestion gel, I calculate that there should be a 1kb band as well.