biochemistry - How does a plant grow before photosynthesis is possible?

There are quite a few questions and thoughts in there, I'll try to cover them all:

First, to correct your initial word equation: During photosynthesis, a plant translates CO₂ and water into O₂ and carbon compounds using energy from light (photons).

You are correct to assume the C is further used for the growing process; it is used to make sugars which store energy in their bonds. That energy is then released when required to power other reactions, which is how a plant lives and grows. C is also incorporated into all the organic molecules in the plant.

Plants require several things to live: CO2, light, water and minerals. If any of those things is missing for a sustained period, growth will suffer. Most molecules in a plant require some carbon, which comes originally from CO₂, and also an assortment of other elements which come from the mineral nutrients in the soil. So the plant is completely reliant on minerals.

Most plants, before a leaf is established or roots develop, grow using energy and nutrients stored in the endosperm and cotyledons of the seed. I whipped up a rough diagram below. Cotyledons are primitive leaves inside the seed. The endosperm is a starchy tissue used only for storage of nutrients and energy. The radicle is the juvenile root. The embryo is the baby plant.

When the seed germinates the embryo elongates, the endosperm depletes, the testa ruptures, and the cotyledons emerge from the seed. The cotyledons are green, and like leaves can photosynthesise, so as soon as they are in the light the plant is able to make carbon compounds. The radicle elongates at the same time, and becomes the root, so the plant is very quickly able to obtain fresh nutrients from the soil (or whatever it's growing in).

At all stages of a plant's life it is using both energy stored in carbon compounds (from CO₂) and nutrients which it took up via its roots. At no point does the plant start to depend solely on the CO₂ in the air for its growth.

You are right that the way in which plants acquire energy and nutrients prior to leaves and roots being established varies between plants. Above I outlined the way most plants use. But there are lots of variations. For example, orchids have very tiny seeds, some barely visible to the naked eye, like specks of dust. They have no endosperm or storage tissue, so they have to rely on a symbiotic mycorrhizal fungus to get carbon and nutrients. The fungus grows through the coat of the orchid seed, then provides everything the growing plant needs until it has its own leaves. Then the orchid repays the fungus by providing sugars.

There are lots of other examples, but we could go on all day!

structural biology - Can protein structure be determined by X-Ray Diffraction in a single image?

Not by analysing a single protein. There is work with x-ray lasers.

You have to take a simultaneous image of millions of proteins and use that to get a structure. It's not quite prime time. People are also doing this with electron beams in electron microscopes.

These methods will reconstruct 3D models of the molecules, sometimes in states which cannot be obtained from crystallography. Examples being the structure of the many megadalton nuclear pore complex, and the f-actin fiber. The classic study is 3d model of bacteriorhodopsin, the first membrane protein structure which was at molecular resolution (this was a crystalline sample though).

While in principle, it sounds much simpler - get a pure sample of your protein, or complex and freeze it down and zap it with an Xray or Electron beam, its a lot more work to reconstruct the image and can take as long or longer than getting an x-ray structure. The resolution is also usually poor as the crystal will reinforce coherence, that is all the proteins are aligned in the same way and have close to the same 3d shape in a crystal.

gel electrophoresis - How do I deal with sticky and viscous samples from cell lysates?

To check a protein expression I pelleted a small amount of E. coli before and after induction and lysed them by redissolving them in SDS-PAGE loading buffer and heating them to 95 °C for 1 minute.

This lead to a solution with some very sticky and viscous parts in it, that make pipetting the sample into the gel wells extremely annoying. As far as I heard, this is probably genomic DNA, and my usual way to deal with this is to centrifuge the samples and only pipet a small part out from the top. This does seem to help sometimes, but not always.

How can I avoid the formation of that sticky and viscious stuff or how can I avoid pipetting that stuff into my wells?

pharmacology - In what ways, if any, does administration of rapamycin *not* mimic calorie restriction?

Rapamycin specifically inhibits the mTOR pathway (mTOR = mammalian target of rapamycin), which has numerous downstream functions including protein biogenesis, regulation of cell cycle, immune function and apoptosis. The upstream effectors of mTOR include growth factors and amino acid availability, so you can certainly see that the lifespan enhancing effects of caloric restriction will be (at least in part) mediated by the mTOR pathway.

But there are key differences. mTOR also receives signals relating to DNA damage and inflammatory changes (to name just 2) that are essential for healthy survival. So any direct inhibition of this pathway will affect all the functions - I can't find the reference now, but I have definitel read in one of the numerous rodent studies that rapamycin treated mice have reduced immune function (i.e. the lifespan increased effects can only be seen in a controlled lab environment - in the wild mTOR-inhibition to this degree would be a disadvantage).

I think therefore it is fair to say that the effects of caloric restriction on longevity are mediated by mTOR, but administration of rapamycin is not an equivalent treatment.

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Update

Really great review came out last month (http://www.ncbi.nlm.nih.gov/pubmed/22500797) - I recommend you give that a skim if you want detail!

human biology - How are proteins formed?

Essentially, yes, "proteins that we consume form new proteins that are different".

The processes are each of them topics for themselves. In short, consumed proteins are digested by peptidases (enzymes) in the stomach, breaking them down into their consituent amino acids. These are absorbed in the gut and transported in the blood to all cells. These take up amino acids and attach them to tRNA molecules which are used in translation to form new body proteins.

human biology - Why do people look different after a long sleep?

What happens during a long sleep that makes people look odd when they have just woken up? Why doesn't the same phenomenon occur in the case of a person who lies down for an extended period of time, but stays awake? I've noticed that some nights seem to make a bigger difference than others in the appearance of the sleeper, but haven't noticed a pattern.

biochemistry - ATP cost for gene expression

The cost of transcribing and translating a hypothetical average gene in yeast has been calculated as 551 activated phosphate bonds ~P per second (Wagner, 2005).

The median length of a yeast RNA molecule is 1,474 nucleotides, and
the median cost of precursor synthesis per nucleotide (derived from
the base composition of yeast-coding regions) is 49.3 ∼P. With a
median mRNA abundance of R = 1.2 mRNA molecules per cell and a median
mRNA decay constant of dR = 5.6 × 10−4 s−1, the mRNA synthesis costs
calculates as 49.3 × 1,474 × 1.2 × (5.6 × 10−4) = 48.8 ∼P per second
and cell. This is a fraction 48.8/1.34 × 107 = 3.6 × 10−6 of the total
RNA synthesis cost per second. The median length of a yeast protein is
385 amino acids, with a combined biosynthesis and polymerization cost
of 30.3 ∼P per amino acid. The median abundance is 2,460 protein
molecules per cell. No currently available data allows a meaningful
estimate of the median protein half-life, but a protein of an
intermediate half-life (see below) of 10 h (decay constant dP = 1.92 ×
10−5 s−1) yields an overall synthesis cost of 30.3 × 385 × 2,460 ×
(1.92 × 10−5) = 551 ∼P s−1.

For your question about a single gene, the cost would be 49.3 * 1474 ~P for the mRNA and 30.3 * 385 ~P for the translation, which would result in around 84 thousand ~P. This is probably a very misleading statistic as you can transcribe multiple proteins from a single mRNA.

How the cost of mRNA synthesis and translation are calculated is described in detail in the paper. A large part of the cost comes from the synthesis of the basic building blocks, the nucleotides and the amino acids.

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human biology - When to measure resting heart rate and blood pressure for following day-to-day trend?

I would be very surprised if the time of day made a difference. I've personally never heard mention of such a phenomenon in discussions with intensive care practitioners (where of course HR and BP are measured constantly). However this is only the case during rest, this paper (on horses) suggests that there is some difference in HR and BP after exercise depending on time of day.

You are right to attempt to control it however, I suppose. It doesn't matter when you take your measurements, as long as you take them at the same time each day.

I would say that one of your first suggestions of taking the measurements almost immediately after waking. Of course you would have to be relaxed as you assembled your equipment then rest for a few minutes in the posture you have chosen (this will matter) before taking the measurements but otherwise I can't see you having too great a problem.

human biology - How reversible is decerebrate posturing caused by brain stem damage?

This is a follow-up question to How likely would Abraham Lincoln be to survive his wounds today?

You don't have to see a CT scan or autopsy to know if the brainstem is
injured (directly or indirectly), if it doesn't work right.

The description of the first doc at the scene mentioned that Lincoln
was not breathing, and one pupil was dilated (the latter a clear and
unequivocal sign of dysfunction of the third cranial nerve or the
upper brainstem, from where it comes). Unfortunately, the second doc
described the enlarged pupil being the right one (it's extremely
unlikely to have been one and then the other - one of the doc's was
probably mistaken as to the side).

By 3 hours after injury, both pupils were fixed and dilated, and
Lincoln showed extensor (decerebrate) posturing - again, all signs of
profound brainstem dysfunction (but not yet brain death, though pretty
close to it).

Now, what is decerebrate posturing? See http://en.wikipedia.org/wiki/Abnormal_posturing#Decerebrate

Decerebrate posturing is also called decerebrate response, decerebrate
rigidity, or extensor posturing. It describes the involuntary
extension of the upper extremities in response to external stimuli. In
decerebrate posturing, the head is arched back, the arms are extended
by the sides, and the legs are extended.[6] A hallmark of decerebrate
posturing is extended elbows.[12] The arms and legs are extended and
rotated internally.[13] The patient is rigid, with the teeth
clenched.[13] The signs can be on just one or the other side of the
body or on both sides, and it may be just in the arms and may be
intermittent.[13]

A person displaying decerebrate posturing in
response to pain gets a score of two in the motor section of the
Glasgow Coma Scale (for adults) and the Pediatric Glasgow Coma Scale
(for infants). Decerebrate posturing indicates brain stem damage,
specifically damage below the level of the red nucleus (e.g.
mid-collicular lesion). It is exhibited by people with lesions or
compression in the midbrain and lesions in the cerebellum

biochemistry - Could hydrogen replace oxygen in cellular respiration?

No, hydrogen could not replace oxygen because it has entirely different characteristics. The most important one is probably its electronegativity - oxygen 'pulls' electrons much 'stronger' than hydrogen.

Basics: Reduction potential

Oxygen is the so-called terminal electron acceptor of the electron transport chain in eukaryotes. You can see "reduction potential" as a kind of stored "energy" which molecules have, similar to the power stored in batteries (very similar actually). To make this text a bit shorter I'll call it "RP" from now on.

One maybe confusing detail is that a substance with low RP has "more energy" than a substance with high RP, so it is the opposite way of thinking.

In very generalised terms, metabolism means that molecules with a low RP (glucose) are oxidised (burned) and turn into molecules with much higher RP (CO₂). Coupled with this, a different molecule with very high RP (oxygen) is reduced and becomes a molecule with slightly lower RP (H₂O). (You may have heard this before - it's called a redox reaction.) *

The important part is that the RP "released" by the oxidisation (burning) is larger than the RP "taken up" by the reduction. The surplus leaves as energy - heat and light if you just burn the glucose. This is a spontaneous process, meaning it will occur just by itself - even if it takes a long time if nobody drops a match on it.

The idea of metabolism is to let that process happen - but use as much of the energy it releases as possible. This works by not just letting it burn, but intercepting that burning process at different stages so that at each step a bit of the RP can be taken off and stored in something else. This "something else" is NAD which I'm sure you've encountered before. Each step that glucose is burned down, another bit of NADH is made, which then has a respectable reduction potential.

NADH (leaving out NADPH here which is a bit different) is channeled into a process called oxidative phosphorylation which retrieves the reduction potential in an actual form of energy.

Basics: Terminal electron acceptor

Finally, the reduction potential I've been talking about all the time is really just electrons, involved in bonds which are "happy" to react. Passing down the reduction potential as I've explained is really a passing down of electrons into more and more stable, less reactive bonds. That's why it's called "electron transport chain". At the end of oxidative phosphorylation, those electrons are dropped onto O₂ and make it into H₂O. That's why O₂ is called the "terminal electron acceptor".

Why Hydrogen can't replace Oxygen

Now to come back to why hydrogen cannot perform oxygen's function in our body. We use glucose as our source of reduction power and oxygen as our terminal electron acceptor. O has a high electronegativity (3.5) so it pulls electrons strongly towards it. H's electronegativity is only 2.1, so it's much weaker. O as a terminal electron acceptor works because it pulls them much stronger than H when they bond, so an O-H bond is almost like giving oxygen an electron. In order for hydrogen gas (H₂) to perform the same function, it would need to be possible to drop electrons onto hydrogen in a bond where it pulls them much stronger than the other partner. They do exist, and such compounds are called hydrides. But the catch is: unlike H₂O, these are normally strong reducing agents, meaning that hydrogen would rather not be in that bond. This not a feasible option for cell respiration, at least in humans, because it requires a lot of RP input. Making oxygen into H₂O does not require a lot, it's a very cheap electron acceptor.

I hope I was able to put this in understandable terms. Let me know if I need to clarify anything.

--

*It works with other molecules than glucose->CO₂ / O₂->H₂O too, many prokaryotes do that and in fact that's how batteries work

t7 promoter - What determines a successful protein expression in E. coli?

I found a very nice paper: Designing Genes for Successful Protein Expression, which covers most factors that determine protein expression. I post parts of it, because I am sure it will be useful to some of you.

Translation can be controlled at the level of initiation and elongation. Initiation of translation is primarily dependent on the sequence of the ribosome binding site (RBS) and early mRNA secondary structure. Other determinants of protein expression are less well understood but equally potent.

1. Initiation of translation

A key component affecting initiation of translation in prokaryotes is the RBS that occurs between 5 and 15 bases upstream of the open reading frame (ORF) AUG start codon. Binding of the ribosome to the Shine–Dalgarno (SD) sequence within the RBS localizes the ribosome to the initiation codon... Affinity of the RBS for the ribosome is a critical factor controlling the efficiency with which new polypeptide chains are initiated. This interaction is in competition with possible base-pairing interactions involving the RBS region that may form within the mRNA itself. Thus, SD sequences with weaker base pairing to the ribosome are more susceptible to interference from mRNA structure. However, some experiments suggest that SD sequences with too strong affinity can be deleterious, particularly at lowering temperatures, by stalling initial elongation. Also critical is the distance between the RBS and the start codon with 5-7 bases from the consensus SD AGGAGG being optimal.

Numerous lines of evidence suggest that the initial 15–25 codons of the ORF deserve special consideration in gene optimization. Studies have shown that the impact of rare codons on translation rate is particularly strong in these first codons, for expression in both Escherichia coli and Saccharomyces cerevisiae. In E. coli, peptidyl-tRNA drop-off during translation of the initial codons appears to be accentuated by the presence of rare NGG codons. These effects appear to be independent of local mRNA secondary structure. It is also true that expression may be recovered by 5' sequence replacement even for sequences that do not show especially strong mRNA structure or contain rare codons or other obvious deleterious elements in this region.

2. Codon bias

The second way in which host codon frequencies can be used is to match the host codon frequencies in the designed gene. This can be done simply by choosing each codon with a probability that matches the host codon frequency...Using sets of genes broadly varied in gene design features, Welch et al. found that variation in synonymous codon usage frequencies had a profound effect on the amount of protein produced in E. coli, independent of local 5’ sequence effects. Variation of at least two orders of magnitude in expression was seen due to substitution beyond the initial 15 codons of the ORF. This variation was strongly correlated with the global codon usage frequencies of the genes, although the codon frequencies found in the highest expressed variants did not correspond to those found in the genome or in highly expressed endogenous genes of E. coli. Multivariate analysis showed that the frequencies of specific codons for about six amino acids could predict the observed differences in expression. It is not clear what the biochemical basis is for this correlation.

3. mRNA structure and translational elongation

While much evidence suggests that mRNA structure can interfere with translational initiation in both prokaryotes and eukaryotes, the effects of structure on elongation are less well understood. This in part may be due to intrinsic helicase activity of ribosomes, which allows translation through even very strong hairpins and may preclude many structures from limiting the translation rate in either prokaryotes or eukaryotes. Perhaps more importantly, mRNA structure is difficult to predict, particularly for actively translated messages which are in continuous flux between various folded and unfolded states.

4. Protein-specific factors providing additional complexity

The protein may be particularly unstable in the host, especially if it is poorly folded due to inherent instability, lack of sufficient prosthetic factors, or improper post-translational modification... Expression of secreted and membrane proteins may be limited by mechanisms for directing these proteins to the membrane. It is even possible that the protein amino acid sequence may limit translational efficiency. For example, proline is thought to be slowly translated in E. coli, regardless of which codon is used.

Expression of the protein may be toxic to the cell leading to instability of the expression vector or host suppression of protein synthesis...A common strategy to reduce toxicity is to lower expression to tolerable levels. Promoters varied in strength can be valuable tools for finding an optimal expression rate for maximal yield...One potential way to avoid toxicity of some proteins is to direct expression to the periplasm or media. This may be accomplished by N-terminal fusion of a secretion signal sequence.

For more information, please read the whole paper. I also recommend reading Design parameters to control synthetic gene expression in Escherichia coli.

genetics - What is the relative power of GWAS studies in different species?

question looks like it's been dormant for a while, but i think there's some discussion to be had here-

I would argue that in many (most?) of the model organisms, power would be much greater than humans. Frequently (worms, mice, plants, yeast) you can work with basically isogenic inbred lines. I would argue this is much more important than long haplotypes:
a) less importantly, no heterozygosity.
b) more importantly, you can re-phenotype the same line repeatedly to directly estimate experimental/environmental variation, and get a very precise estimate of actual E(phenotype | genotype). this is opposed to humans, where you have the one individual, so you just have to pray that your experimental/environmental variation is low and your heritability is high in the sample population.

For example, for just 100 inbred lines of a plant, you get massive, beautiful GWAS peaks for many phenotypes: Atwell et al. 2010. Human GWAS sample sizes generally have to be in the 1000s before they are sufficiently powered (ref).

This subject is discussed a little further here.

In direct reference to the long haplotypes, note that power and mapping precision are different things. That is, your power to detect an association can be extremely high, but you might have a very broad chromosome interval that you will then have to go hunting in to find the causal locus. Note that this problem might actually be worse with large effects, which are probably under selection if they are interesting, and thus there is likely to be substantial linkage disequilibrium between the causal locus and surrounding regions. Of course, if the effect size is small (generally the case in humans though not in other orgs), this is less of an issue.

pharmacology - Is it better to take a half dose of paracetamol and a half dose of ibuprofen together rather than a full dose of either?

Whilst Alexander Galkin gives some great information, I think there's a fundamental reason why that particular pain relief strategy is best, and it hasn't been mentioned yet.

The reason is simply that when you take Ibuprofen or Paracetamol (a full dose) you can only take it once every 4 hours. However, the pain relief doesn't last for 4 hours, so if you take either or both together every four hours you will experience pain after the medication wears off and before you are allowed your next dose.

So instead you can follow either of two strategies:

1. Take a full dose of Paracetamol at 0 hours, 4 hours, 8 hours, as well as taking a full dose of Ibuprofen at 2 hours, 6 hours, 10 hours. This will give you less time when no pain medication is in effect. But if your pain is inflammation related you might want to keep the anti-inflammatory effects of Ibuprofen topped up, so you can...


2. Take a half dose of Paracetamol + a half dose of Ibruprofen every 2hours. This gives you a mix of pure pain relief and anti-inflammation and ensures minimum time when each medication has no effect.

I don't think human biology has to come into it (apart from the fact that you can take the two substances together with no ill effects), it's just a good pain-avoidance strategy.

human biology - How exactly does long-term tension (over months) build up in the ligaments, tendons, muscles?

Tension is not a quantifiable thing, rather an abstract concept to describe feelings of stress in muscles. I think I see what you are getting at but I would separate it into two questions.

"Is massage provably beneficial to muscles under physically induced stress?"

Emphatically yes, however there are so many different types of massage, some are more medically viable than others. Most massage stimulates muscles to encourage a release of tightening, much like exercise does, though I would assume that there's also a psychosomatic that plays a significant role as well due to human contact.

The other question, more the point "Are massage therapist's claims about relief true?"

Some are and some aren't. Unless it's by a medical professional (prescribed physical therapy for instance) they can claim whatever they like. "Reiki contributes to your body balance and wellness by attuning your energy" This can not be proven, but not really disproven either, so they can claim it all they want though it's purely anecdotal (if not outright fabrication on the part of less ethical individuals.)

In summary: Massage has tangible benefits, but if the claims of the therapist sound too good to be true, they probably are.

biochemistry - When collecting cell lysates for a Western blot, how do I induce di-sulfide bonds?

I would like to conduct a simple dimerization experiment for some protein I'm collecting from a cultured cells. My thought is, that if I'm running a non-reducing, denaturing PAGE gel, then removing beta-mercaptoethanol/DTT from the sample buffer should be enough to allow di-sulfide bonds to form.

I have seen several authors incubate the cells first with a drug called BSS that diffuses across membranes and creates protein cross-links. I may be wrong, but the use of this drug seems more appropriate when trying to follow up with an immunoprecipitation or a pull-down assay.

If anyone has any experience with this, could you please enlighten me?

What is special about the N3 buffer for Qiagen minipreps?

N3 and P3 are different. N3 contain chaotropic agents. P3 does not.
Mainly there are two types of DNA purification kits on the market: silica membrane (glass fiber) based and DEAE anion exchange based. The silica membrane based method requires high concentration of chaotropic agents (salts that can change the water structure dramatically, GuHcl, GuSCN, NaI, are a few of them) present in the mixture before loading on to the spin column. DEAE based method, on the other hand, bind DNA based on the charge property on the DEAE resin which largely depends on the pH and ionic strength of the buffer. Chaotropic agents are not needed for DEAE based method.
In our lab we make our own kit using components and buffer recipe provided by Epoch Life Science for both mini prep and maxi prep. Their mini spin column and maxi DEAE column can be purchased without buffer. Their mini spin column also works perfectly with buffer from Qiagen and Lifetech. That's how we used up our leftover buffer from our old kits.

pharmacology - Looking for a cancer drug target database to guide sequencing of patient tumor DNA

I have a question I would like to pose to the community. I have recently received access to a bench-top ion torrent DNA sequencer. Our idea is to use this machine to sequence the DNA from patient’s tumors in order to guide treatment options. My job is to identify a list of all currently used anti-neoplastic drugs along with their known targets (i.e., specific genes and mutations) and accession numbers. I would like to put these data in a table in which each row corresponds to a different drug.

For example, a row in the table might read (column names are indicated in brackets): [disease] breast cancer, [drug] trastuzumab, [drug target] HER2/neu receptor, [gene] ERBB2, [location] chr17:37844393-37884915, [mutation type] amplification, [accession number] ENSG00000141736. The pathologists would then be able to use this database in order to select appropriate genes for sequencing whenever they receive a tumor specimen. If the patient’s tumor had an amplified ERBB2 gene, they could be given trastuzumab.

Currently our study is in pre-planning stages (i.e., we won’t actually be testing this on patients any time soon). I would appreciate it if anyone could give me on advice on how to go about creating such a database. I am aware of online databases including COSMIC, Sanger's Cancer Gene Census, and the Potential Drug Target Database (PDTD), but they don’t have everything that I’m looking for. I am familiar with R and could use it to combine data from multiple sources if necessary. If anyone else has comments or suggestions for further reading that would also be appreciated. Thanks!

Note: This question has also been posed on a Research Gate forum: http://www.researchgate.net/topic/Cancer_Biology/post/Looking_for_a_cancer_drug_target_database_to_guide_sequencing_of_patient_tumor_DNA

genetics - Relative Property of Alleles

Sure it is!

When we say that an allele is dominant on another what we really mean is that the phenotype obtained from that specific mutation of the gene somehow "masks" the phenotype of the other.

The easiest example would be that of pigment. A gene X could code for an enzyme which produces a red pigment. A mutation in X could result in a allelic variant x coding for a non-functional enzyme that does not produce pigment. In this case X will be dominant on x, as in the presence of both the pigment will still be produced (because you will have half functional and half non-functional enzyme molecules).

Things can, however be slightly complicated.

For instance, if the allele R codes gives a red pigment and r gives a white pigment Rr could give pink. This is called incomplete dominance.

On the other hand you could have co-dominance, where WW is white, ww is black and Ww is black and white.

The example you are asking for is called serial dominance and the "classical" example given is that of the rabbit coat color where you have for alleles with a dominance hierarchy like:

c+ > c^ch > c^h > c

c+ gives a fully functional enzyme, called tyrosinase
c gives a non-functional enzyme, resulting in an albino phenotype
c^ch (chincilla phenotype) and c^h (hymalayan phenotype) give partly functional enzyme (interestingly, the latter gives a temperature sensitive fur color).

In reality the rabbit coat genetics is even more complex, see this documents (PDF) for a more complete list of all the various loci and possible alleles involved.

population biology - What's in a Name: Statistical Genetics

Mendel published his results 1866 but they were rediscovered only in 1900. The Hardy-Weinberg model is an application of Mendel's rules to a population that is not under selection forces. So the one builds on the other, and Hardy-Weinberg is a simplification model-wise, and Mendel's rules are not detailed enough either. It's the same relation as with a physical law like gravitation and a mathematical model of its consequences applied on many entities, like models of a solar system's gravitational behaviour.

botany - Why do some fruits have a much wider range of acceptable sizes than others?

Pumpkins, squashes in general, grow on vines, while apples grow on trees. Vines are fast growing and trees are not. Zucchini can be quite large; cucumbers, too. Pears, plums, peaches and other tree fruits do have a reduced variation in fruit size. While I do not know the answer to your question, my background in plant biology tells me that this is an important part of the reason. For example, a watermelon or pumpkin rests on the earth, but a tree fruit hangs. If that fruit is too heavy it falls from the ground before it is ripe - before the seeds are fully differentiated that is. So, while "evolution" might be an answer, it is important to discuss what aspects are at play.

I think it has to do with vine vs tree, and how the fruit is attached and hangs/lays.

zoology - Effect of spaying on the female cat organism and health

From the standpoint of the skeletal system, spaying is definitely not good for her health. Removal of the ovaries mimics what happens at menopause, when circulating estrogen levels fall. Estrogen is necessary for maintenance of bone mineralization. Without estrogen, bones start to demineralize (the same process that happens in human females after menopause, leading to osteoporosis).

Then again, some animals, if they do not get pregnant, will cycle indefinitely until they get anemic and die. Ferrets are an example. So it's better to have these spayed.

evolution - Do large animals often evolve into smaller animals?

Your question brings up several important issues with regards to the evolution of body size. The rationale for concluding that the ancestral mammal had a small body size is that all of the taxa in that area of the tree tend to be small. In contrast, if all of those taxa had been cow-sized, then the most parsimonious conclusion would be that the ancestral mammal was cow-sized.

Identification of ancestors is difficult

Identification of a fossil species as the most recent common ancestor of a pair of sister taxa is exceedingly difficult. The way that different species are diagnosed in fossils in by their unique derived characteristics (autapomorphies). So if a fossil is found with no autapomorphies, then it is plausibly an ancestor (i.e., at the split between two sister taxa). Some would argue that if you don't find any autapomorphies, then you just haven't looked closely enough.

Cope's Rule

The idea that lineages tend to evolve toward larger body size is known as Cope's Rule, named for the paleontologist Edward Drinker Cope. The success of Cope's Rule has been highly variable, with some lineages following and others not (Polly, 1998).

Some clades following Cope's Rule

Some clades not following Cope's Rule

Note that many results depend on the methods used.

Evidence from experimental evolution

Several studies have addressed body size evolution in vertebrates both directly and indirectly. MacArthur (1944a, 1944b) directly selected for large and small body mass in laboratory mice. Within 8 generations, mean body masses had increased or decreased by over 50%. Even when body mass is not directly selected upon, mass can change in a correlated way. Selection for high levels of voluntary activity has led to an approximately 25% reduction in body mass.

Based on these studies, evolution of body size (larger or smaller) is certainly possible within relatively few generations.

pathology - Why are some bodily fluids more of an infection risk than others?

This is just about where the pathogens can be found that are dangerous to people.

Vomit is highly acidic and less accommodating to microbe growth. Similarly saliva has many immune components in it as well as digestive enzymes that keep most microorganisms down.

Urine and CSF are actually quite sterile as they come from environments that are highly filtered - the kidney is an osmotic processor that essentially is a molecular filter and does not allow cells to pass, the spine is highly insulated from the blood and other direct exposure to microorganisms.

Compare that with the 'dangerous' list and you have organs that are open to human pathogens. Venerial disease like HPV is so common that what - about 1 in 5 people under a certain age carry it. That is a pretty high expectation of a biohazard. most infections and viruses are blood bourne - influenza, cold, as well as any bacterial infections.

Feces is always a dangerous thing to handle as the digestive tract is rich in nutrients and essentially directly open to external bacteria and fungi. (and its not acidified like the stomach). Also parasites like tape worms and other multicelled animals! yum!

Diarrhea is often caused by an infection of some sort, so its just more likely a hazard, but feces is always a place where you might find a pathogen.

This is not to say that the 'safe' list is totally safe. Its just less likely to bear disease causing agents.

zoology - Can all mammals swim?

With respect to the giraffe claim, this article seems relevant:

D. M. Henderson, D. Naish, Predicting the buoyancy, equilibrium and potential swimming ability of giraffes by computational analysis, J Theoretical Biology 265 (2010) 151-159.

It cites several non-"random person on the internet" claims that giraffes cannot swim:

It is generally thought that giraffes cannot swim, but relevant
observations are few. Shortridge (1934) and Goodwin (1954) state that
giraffes were poor waders and unable to swim. Crandall (1964)
discussed a case where a captive giraffe escaped from a carrying
crate, ran to the end of a jetty, and fell into the water. The animal
reportedly sank without making any attempt to swim. MacClintock (1973,
p. 54) stated ‘Giraffes cannot swim. Rivers are barriers they do not
cross’. Wood (1982, p. 20) noted that ‘Because of its extraordinarily
anatomical shape the giraffe is one of the very few mammals that
cannot swim – even in an emergency! Deep rivers are an impassable
barrier to them, and they will avoid large expanses of water like the
plague’.

They then go on to show that a model giraffe could plausibly swim, writing: "For practical and ethical reasons we are unable to use live giraffes..."

They conclude:

In summary, the results and speculations of this study show that it is
not impossible that a giraffe could propel itself in water, but in
terms of energy efficiency relative to that of the horse, it would
appear that the costs of aquatic locomotion might be too high. It is
reasonable to expect that giraffes would be hesitant to enter water
knowing that they would be at a decided disadvantage compared to being
on solid ground.

immunology - Do you know of any disease where HLA / MHC association discovery has led to better treatment or management?

If we discover an association between a disease and a particular HLA type (MHC class I or class II molecule) then it may provide us with some insight into the disease in a very basic way. However, will it provide us with a therapeutic target? Or second best, will it provide us with information that is useful for diagnosis or management? I am looking for examples. Do you know of any?

The most obvious is in transplantation where one would use HLA typing to match donor to recipient but that is not the same as using knowledge of an HLA type to target, treat, diagnose, prognosticate.

gel electrophoresis - What is the DNA/protein charge ratio?

This is really sounds like a guideline, a starting place, not necessarily your best protocol.

In practice its hard to be more specific as MS says. The pH of the buffer will affect the charge of the protein, and to a lesser extent the DNA.

Also the protein may have an ideal pH for binding which you could guess is in the range of 7.0-7.4, but might find that the strongest binding depends on unexpected pH and salt concentrations, or the addition of a small molecule.

The affinity of araC for instance is greatly increased by the presence of arabinose in sufficient concentration.

lac Repressor, in contrast releases itself from DNA in the presence of IPTG or lactose.

evolution - Could an organism theoretically produce a metamaterial-like structure?

I'm curious to know if this is physically feasible because during my reading up on synthetic biology and just general research i realise that life is capable of producing some exquisitely complex materials and biological structures and wondered if they could potentially recreate artficial materials that aren't found in nature well not yet anyway. Could such a structure evolve naturally or would it have to be designed or directed via synthetic biology techniques?

Thank you for your time.

eyes - Are rods/cones rejuvenated periodically?

Pretty much what the title states.

Some cells/structures in the human body undergo rejuvenation periodically by flushing away older cells and growth of new ones.
E.g Skin, Blood

Does this (for want of a better word) 'rejuvenation' apply to Rods/Cones within the eye too? If so, how are the old cells flushed out - or do the old cells become part of the local humour?

bioinformatics - What is the significance and method behind Ramachandran plots?

Ramachandran plots show the relationship between the phi and psi angles of a protein referring to dihedral angles between the N and the C-alpha and the C-alpha and the C-beta. As an aside, the omega angle between the C-beta and the N tends to be fixed due to pi-pi interactions.

Dihedral Angles

There are limits to possible distributions of phi and psi angles due to steric clashes between the side chains. Furthermore other limitations from higher order structure will result in the adoption of defined phi-psi angles. Using data from solved crystal structures, it can be seen that the dihedral angles will adopt specific conformations in a protein.

RAMACHANDRAN PLOT from Lovell et al.

Furthermore, it can be noted that some of these conformations relate to specific secondary structures. As seen above, peptides in alpha-helices and beta-sheets adopt a even more limited set of phi-psi angles. Certain amino acids like glycine and proline, which differ from from canonical amino acids have an unique Ramachandran plot.

The angles from a Ramachandran plot are useful not only for determining a amino acids' role in secondary structure but can also be used to verify the solution to a crystal structure. Furthermore, it assists with constraining structure prediction simulations and helps with defining energy functions.

homework - Does Human Female Meiosis II occur after fertilization with sperm?

Meiosis, as you know, have two stages, Meiosis I and II. The oocyte is arrested during metaphase II of MEOISIS II. This arrest is facilitated by a complex called "Cytostatic Factor" (CSF).

After fertilization, the sperm induces a rise in intracellular calcium ion which activates and enzyme, Calmodulin Kinase II. This complex, through a series of phosphorylation and ubiquitination, degrades the CSF comples and in turn, activates APC (Anaphase Promoting Complex). APC will then degrades cyclins, securins and this will promotes the completion of Meiois II.

Sperm must ignite some process in female that puts female meiosis II going on before sperm can fuse with egg.

I think the statement is a bit incorrect. The sperm will ignite (I prefer induce) the above changes and fuse at the same time. Because, what starts all the process above is part of the sperm's cytoplasm that need to be assimilated into the oocyte's cytoplasm.

I hope this clears it all up. Or if you are interested, I can provide the long list of signalling pathways that leads to zygotic development.

genomics - Which functional annotations could be useful?

Analyzing a genome, for a generic gene, which functional annotations (e.g from Gene Ontology) can help understanding its meaning/function or, at least, provide helpful informations?

• Annotations of that gene


• Annotations of orthologous genes (?)


• Annotations of paralogous genes (?)


• Co-expressed genes (given a biological condition) (?)


• What else?

Thank you.

evolution - Did animals evolve from plants?

See this paper "Divergence time estimates for the early history of animal phyla and the origin of plants, animals and fungi" for information on the divergence estimates (I'm not sure if there are more recent papers discussing this).

Plants, animals and fungi are eukaryotes, distinct from eubacteria and archaebacteria, which are prokaryotes. The difference being in the composition of the cell, particularly a nucleus contained within a membrane for eukaryotes, along with other membrane bound organelles, e.g. chloroplasts. They all share a common ancestor, according to this paper, that split 1.576 Bya (billion years ago) +/- 88 Mya (although it states the relationships are unresolved - it is often difficult to resolve relationships so deep in a tree). They form distinct groups known as Kingdoms under Linnaean based biological classification; the Fungi, Plantae and Animalia. Thus, in answer to your question, no, animals did not evolve from plants.

Plants have chloroplasts in their cells, which provide the ability to produce energy via photosynthesis. It is thought that the chloroplast resulted from a symbiotic relationship between early plants and a cyanobacteria in that they both relied on each other for survival and so coevolved. Animals don't contain chloroplasts and instead contain an organelle called the mitochondria (although most plants also have mitochondria), which is also thought to have been a bacterial endosymbiont, probably related to rikettsias.

Protists also contain chloroplasts. The protists are intermediate between all three groups and have been notoriously difficult to classify, being placed into a fourth Kingdom, the Protozoa, although this grouping has been contested. The current Cavalier-Smith system was proposed in 2004 and classifies life into 6 Kingdoms.

Chloroplasts are thought to have evolved from a single endosymbiotic event in Archaeplastida, although there are evidence to suggest some secondary endosymbiotic events. Check out this paper for more information; figure 1 shows the relationships between the different groups and the endosymbiotic events. The Opisthokonts are the origin of the Fungi and Animalia kingdoms.

human biology - Why Does Salt Water Help Sore Throats?

Salt water may have anti-septic properties due to the effect it has on water potential. Pure water has a water potential (Ψ) of zero. A concentrated salt solution has a lower (more-negative) water potential. The water potential of the salt solution is likely to be more negative than that of the pathogen's cytoplasm; the salt solution is therefore referred to as hypertonic. Therefore water osmoses out of the cell (osmosis being the net movement of water from a higher water potential to a lower water potential across a semi-permeable membrane). The loss of water from the pathogenic cells causes osmotic crenation - the cell becomes shrivelled and dies.

A hypotonic solution (for example cells placed into pure water) would cause the opposite effect - osmotic lysis. This is the bursting of the cell due to the movement of water into the cell. The bacterial cell wall would first have to be damaged (e.g. by penicillin). This would not be the process by which a salt solution has effect, however.

The fact that the salt water is warm in order to improve solubility may also have the side-effect of causing vasodilation around the infection, increasing the rate at which white blood cells can arrive at the infection site.

It has been more difficult to find a theory as to why a salt solution would have analgesic properties, see the comments below & previous versions of this answer.

genomics - Is it possible to trace of the ancestry of a person by only using his/her genetic information?

In short, yes, it is possible. There are companies that sequence part of your genome and then can trace it back to your ancestors. All human family trees can be traced back to their African origin 200 000 years ago, but the companies that sequence your genes do not do that. For example, 23andMe sequences only 1 million of your base pairs (single nucleotide polymorphisms (SNPs)). They trace your most recent heritage, which could lead to Europe, Africa, or Asia. They can basically trace which part of your chromosomes (and thus your ancestry) came from your mom or your dad:

Unlike the sex chromosomes and the mitochondrial DNA, which are inherited as blocks, the 22 biparental chromosomes, known as autosomes, are scrambled during reproduction. Through a process known as recombination, each parent pulls his or her paired set of 22 autosomes into chunks, then reassembles a new single set using half the material from each pair. The two single sets of chromomes from each parent are combined into a new paired set when a sperm fertilizes an egg.

You can read more about 23andMe here. There are other companies that are specialized in tracing your ancestry: Genetic Genealogy, DNA Tribes, and Heirlines.

genetics - Predicting progeny of recessive mutations using recombination

First, the recombination equation is:

cM = recombinants/(recombinants AND parentals) * 100

Let's assign the following genotypes for clarity's sake:

RR = black bristles
Rr = black bristles
rr = red bristles
SS = pebbly eyes
Ss = pebbly eyes
ss = shiny eyes

F1 cross: rrSS x RRss = RrSs (all progeny)
F2 cross: RrSs (F1) x rrss = ?

You would have the following gametes:

Parent 1, RrSs = RS, Rs, rS, rs
Parent 2, rrss = rs, rs, rs, rs

Draw your punnet square and you get the following genotypes of the offspring.

So your GENOTYPES of your F2 cross will be RrSs, Rrss, rrSs, rrss

Let's translate those to phenotypes.

RrSs: black pebbly (parental)
Rrss: black shiny (recombinant)
rrSs: red pebbly (recombinant)
rrss: red shiny (parental)

Now let's look at recombination frequency.

15cM = (recombinants/(recombinants+parentals)*100
recombinants/(parentals+recombinants) = 15/100

This tells you for every 15 recombinants, you have 85 parentals. If you scale this up to 1000, that's 150 recombinants for every 850 parentals.

Your 150 recombinants comes from 75 black/shiny and 75 red/pebbly.

Your 850 parentals ceoms from 425 black/pebbly and 425 red/shiny.

Anyways that was my attempt which appears to be directly opposite to what your answer key says. Did you copy it down correctly?

lab techniques - What effect does vortexing have on a fluid sample that simple mechanical shaking does not?

what specific effect the vortexing has that makes it better than manual shaking

In addition to @bobthejoe's answer about viscous fluids, (manual) mechanical shaking is also less consistent and more tiresome than vortexing.

If the vortexer is always at the same speed and each sample is vortexed for the same amount of time, then the shaking step will be less variable among samples. For example, the vigor of mixing an aqueous soil suspension could potentially affect the amount of aggregate decomposition and release of molecules into the suspension (I don't have a reference for this, but it is good practice to treat replicates the same to minimize any non-treatment effect).

Manual shaking is not only less consistent, it is also tiresome - especially with larger volumes (>=10ml) and large sample sizes.

biochemistry - Does GFAJ-1 use Adenosine triarsenate as its energy currency?

This is a cool topic/question.

To answer your question. The hypothesis was based on the conjecture that there was so little phosphorus in the culture medium that phosphorous would have been replaced by arsenic in all its roles in the cell. IF they had found arsenate DNA, it would have been derived from NTAs (nucleotide tri-arsenates) or a hybrid Phosphorous/Arsenic analog of the compound as DNA polymerase consumes NTPs to create DNA. If there were only NTAs to drive DNA biosynthesis, then the cell's energy cycle would also have had to use ATA.

BUT

The primary evidence was that the mono lake strain grew in a fermentor (culture) with lots of arsenic (which is impressive) and very little phosphorous. how little? 3 micromolar. The investigators say that they did add a little phosphorus (3-5 micromolar), which, after some more careful accounting, appears to be enough to keep the bacteria growing at the observed rate without using arsenate nucleotides (submitted to Science).

This is not completely surprising as the original publication in 2010 of a preliminary finding in Science Express which only had x-ray abosorbtion fine edge spectroscopy work consistant with an arsenate like that found in a phosphorus backbone. Given that they did not produce a more direct reading of the compounds such as mass spec or an NMR experiment, this looked pretty iffy in the first place.

You an see why arsenic life was so improbable - a dozen (or more) vital pathways in the cell would have to adapt to use NTAs - pretty much all at once. If they had I suspect Mono lake would be full of those suckers.

Its sort of a bummer, for those of us who want to discover new forms of life, but you can't find what isn't there.

cell biology - Intrinsic apoptosis in erythrocytes

Red blood cells (RBCs) don't have nuclei in mammals, so they are usually considered to be pretty inert compared to other cells.

They do have some biochemical activity, and evidently there is some mechanism for cell death in RBCs when they are induced to degrade or infected by pathogens. I found this reference that shows caspases -8 and -3 are evident in mature RBCs. Several other components of the cell death pathway are not evident.

At this time the authors speculate that they don't activate to induce cell death.

It seems that over the broad field of disease research 'apoptosis' and 'cell death' are used equivalently even when the well known apoptosis pathway is not involved.

nutrition - Human Body "Fat" Equilibrium

I'm not quite sure what you mean by

regardless of x and y being out of balance.

Surely it would make more sense to assume you gain 0 weight if x and y are balanced? The prime reason why people get obese is probably because x and y are not balanced for them (there are other reasons though, as well as reasons why people can have it imbalanced and still not gain weight).

1. Even if you consume exactly as many calories as you expend in a day, you may gain weight depending on how (ratio of protein/fats/carbohydrates) and especially when you consume the calories. The body may for example store consumed amounts as fat after eating and then use carbohydrate storage from liver and muscle when starving, so long term you would still gain weight.



2. Adipose (fatty) tissue requires energy too. If you consume in the same manner after as before cutting a large amount of it away, you may just regain it until x=y again.




3. Also consider: if people ate just half an apple a day more than the exact amount of energy they need, they should gain more than a kilo on average every year. But they don't, and you can hardly assume that people manage on average to consume the exact amount their body needs.

The reason for that is probably leptin. It is a hormone produced by adipocytes (fat cells) and essentially indirectly reduces food uptake and increases expenditure, among others by decreasing your appetite. This has been regarded as a sort of "adipostat"; your leptin system creates a genetically determined level of body fat that you will tend to obtain. Dieting or cutting away fat will cause a drift to return to the previous weight because of a lower level of leptin (due to reduced fat cells). Of course it's not as simple as that though, so it's also possible that you will actually long-term reduce weight through those methods. E.g. exercise seems likely to be an exception somehow.

biochemistry - How to compute properties of peptides ?

I have been tasked with writing a program for computing properties of a give set of peptides. These peptides are given as 1-letter amino acid sequences and I need to compute the following :

• Length of peptide


• Number of Each Amino Acid


• Percent composition of each amino acid


• Molecular weight


• Net charge of peptide


• Positive charge


• Negative charge


• Isoelectric point (pI)


• Hydropathicity


• Percent polar amino acids


• Percent positive amino acids


• Percent negative amino acids


• Percent hydrophobic amino acids


• Hydrophobicity


• Lipophilicity


• Amphiphilicity


• Water-Octanol Partition Coefficient


• Steric Bulk


• Side chain bulk


• Net donated hydrogen bonds


• Percent alpha helix


• Percent random coil


• Percent beta sheet

While some of these properties are self explanatory ( eg. size, num. of amino acids, percentage of amino acids. ) and easy to compute. Other properties ( like Molecular weight, Net. charge, Positive charge, Hydorphobicity etc ) have been difficult for me.

I donot have Chemistry or Biology background and hence have found these difficult to compute. I would be appreciative if someone could point me in the correct direction ( I have already been through Wikipedia ) containing methods to compute the above mentioned properties or to a standard text which would explain the above mentioned properties and also provide methods to compute them. Thank you all.

abiogenesis - Why are amino acids in biology homochiral?

I know that you are referring to the commonly ribosome-translated L-proteins, but I can't help but add that there are some peptides, called nonribosomal peptides, which are not dependent on the mRNA and can incorporate D-amino acids. They have very important pharmaceutical properties. I recommend this (1) review article if you are interested in the subject. It is also worth mentioning that D-alanine and D-glutamine are incorporated into the peptidoglycane of bacteria.

I read several papers (2, 3, 4) that discuss the problem of chirality but all of them conclude that there is no apparent reason why we live in the L-world. The L-amino acids should not have chemical advantages over the D-amino acids, as biocs already pointed out.

Reasons for the occurrence of the twenty coded protein amino acids (2) has an informative and interesting outline. This is the paragraph on the topic of chirality:

This is related to the question of the origin of optical
activity in living organisms on which there is a very
large literature (Bonner 1972; Norden 1978; Brack and
Spack 1980). We do not propose to deal with this
question here, except to note that arguments presented
in this paper would apply to organisms constructed from
either D or L amino acids.

It might be possible that both L and D lives were present (L/D-amino acids, L/D-enzymes recognizing L/D-substrates), but, by random chance the L-world outcompeted the D-world.

I also found the same question in a forum where one of the answers seems intriguing. I cannot comment on the reliability of the answer, but hopefully someone will have the expertise to do so:

One, our galaxy has a chiral spin and a magnetic orientation, which causes cosmic dust particles to polarize starlight as circularly polarized in one direction only. This circularly polarized light degrades D enantiomers of amino acids more than L enantiomers, and this effect is clear when analyzing the amino acids found on comets and meteors. This explains why, at least in the milky way, L enantiomers are preferred.

Two, although gravity, electromagnetism, and the strong nuclear force are achiral, the weak nuclear force (radioactive decay) is chiral. During beta decay, the emitted electrons preferentially favor one kind of spin. That's right, the parity of the universe is not conserved in nuclear decay. These chiral electrons once again preferrentially degrade D amino acids vs. L amino acids.

Thus due to the chirality of sunlight and the chirality of nuclear radiation, L amino acids are the more stable enantiomers and therefore are favored for abiogenesis.

1. BIOSYNTHESIS OF NONRIBOSOMAL PEPTIDES




2. Reasons for the occurrence of the twenty coded protein amino acids




3. Molecular Basis for Chiral Selection in RNA Aminoacylation




4. How nature deals with stereoisomers




5. The adaptation of diastereomeric S-prolyl dipeptide derivatives to the quantitative estimation of R- and S-leucine enantiomers. Bonner WA, 1972




6. The asymmetry of life. Nordén B, 1978




7. Beta-Structures of polypeptides with L- and D-residues. Part III. Experimental evidences for enrichment in enantiomer. Brack A, Spach G, 1980

visualization - How to typeset gene regulatory networks

I have a relatively simply gene regulatory network I would like to visualize, complete with the common arrow and bar symbols used to show, respectively, which genes enhance or repress with other genes. Is there a way to typeset a gene regulatory network using something like LaTeX or Graphviz?

For very simple networks, I guess something like Photoshop or Illustrator would probably be the quickest solution, but these programs become very tedious as the size of the network grows even a little bit. What do people typically use for this type of task?

cell biology - Regarding TIMP and MMP enzymes

For your first question

Both enzymes, are components of Epidermal growth factor receptor (EGFR) signalling pathway.

MMP is a matrix metalloproteinase, that triggers EGFR signalling, this can be done by a mechanism called transactivation

So, A signal from G-coupled receptor, or cytokine receptor could activate the cleavage of MMP, then MMP converts to a ligand for EGFR activation

For example Angiotensin II type 1 receptor (AT1R) and type 2 receptor (AT2R) which are GPCR, there are evidence for crosstalk with EGFR, look for this paper. This could be linked with hypertension or inflamation. Also cancer, could trigger EGFR receptors by NMP's.

And finally tissue inhibitors of metalloproteinases (TIMPs) are the negative regulator of MMP's

This are only an overview of the whole biological process involving MMP, so I think there is no single answer to your question, but you can delimit the topic for a specific disease, and look for papers at google scholar.

biochemistry - Can siRNA induce DNA methylation in mammalian cells?

Some years ago Hiroaki Kawasaki and Kazunari Taira published an article called "Induction of DNA methylation and gene silencing by short interfering RNAs in human cells" in Nature:

In plants, dsRNAs targeted to CpG islands within a promoter can also
induce RNA-directed DNA methylation^{3, 4, 5, 6, 7, 8}; however, it
remains unclear whether gene silencing mediated by DNA methylation can
be induced by dsRNAs in mammalian cells. Here, we demonstrate that
short interfering RNAs (siRNAs; 21–25-nucleotide RNA molecules) induce
DNA methylation and histone H3 methylation in human cells.

This paper seemed to show that siRNA can also induce long-term silencing of genes by DNA methylation in mammalian cells, which hasn't previously been observed. Unfortunately, the paper was retracted later, calling the results into question.

I found the paper very exciting at the time, as it suggested the possibility to perform gene therapy, to silence specficic genes, just by using RNA interference.

What is the current scientific consensus here, is there any convincing evidence that siRNA-induced DNA methylation is possible in mammalian cells? Was anyone able to replicate the results from Kawasaki and Taira?

biochemistry - What happens to dextrorotatory amino acids in humans?

You may also be interested in D-amino-acid oxidase (EC 1.4.3.3), a flavoprotein (FAD) highly specific for the D-form of amino acids, which was discovered by Hans Krebs in 1935 (see here), and which has a wide distribution (including in humans).

The enzyme has been very thoroughly investigated, in particular by Massey & co-workers (see here for example)

D-amino-acid oxidase (EC 1.4.3.3) catalyzes the reaction which results in the following transformation

D-amino acid + H₂O + O₂ = 2-oxo carboxylate + NH₃ + H₂O₂

2-Oxo-carboxylates are what used to be called α-keto acids. For example, pyruvate is produced from D-Ala.

The product of the enzymatic reaction is the imino-acid which is nonenzymatically
hydrolyzed to a-keto acid (see Pollegioni et al., 1994, and references therein)

An excellent review

• D-Amino Acid Oxidase: Physiological Role and Applications

  
  
  

  by S. V. Khoronenkova & V. I. Tishkov,

  
  
  

  Biochemistry (Moscow) is freely available from here

These authors have some intersting things to say about D-Serine, D-Proline and D-Alanine, and much more.

Additional Reference

• Pollegioni L, Fukui K, Massey V. (1994) Studies on the kinetic mechanism of pig kidney D-amino acid oxidase by site-directed mutagenesis of tyrosine 224 and tyrosine 228. J. Biol. Chem. 269, 31666-31673. [pdf]

synthetic biology - DNA synthesis companies: cost per base, turn-around time, codon-optimization algorithms

Are you working in academia? It might be worthwhile to call the companies directly and see if there is a discounted rate for your institution (another lab or department may have already set something up).

You won't be able to get information on the different algorithms if they are proprietary. However considering that codon usage data is public and shared knowledge, I can't imagine that the different algorithms are that much different. I think there are some tricks for choosing certain codons near the transcription start site in some organisms? From what I hear, the algorithms are constantly changing. I could tell you how my E. coli optimized gene from GenScript worked two years ago, but they'd probably design it differently now.

Okay - GenScript calls their algorithm "proprietary", so they are not sharing the details. DNA2.0 says their algorithm is patented, so it's out there for inspection. They published a PLOS paper on their E. coli algorithm: Welch et al..

Although, maybe all they do these days is spit out codon sequence randomly:

The results obtained in this study indicate that the codon
randomization method is a superior strategy for codon optimization. A
significant improvement in protein expression was obtained for the
largely established process of chymosin production, showing the power
of this strategy to reduce production costs of industrial enzymes in
microbial hosts. (Menzella 2011)

Stitching smaller synthesized fragments works well if you're pressed for time, but it's usually more expensive. However, sometimes these companies do special pricing on synthesis for <1kb, which makes stitching a great choice. Turnaround is generally faster for three 1kb fragments than one 3kb fragment.

bacteriology - Are the intestinal microbiota substantially different within a diverticulum of the colon?

The appendix is a type of true diverticulum of the colon, although it is one that is present in everyone (unless a surgeon has removed it). There are bacteria in the gut from the mouth all the way down to the anus, and the numbers and types of bacteria change along the length of the intestinal tract. A recent review from J. Ridlon published in the Journal of Lipid Research (2006) is available online and has documented these observations from a number of investigators. In that paper the bacterial species are described as changing as well as the relative abundances of bacteria which increases along the length of the intestinal tract. Figure 1 from that paper which is open access available from the link above is below.

I think its interesting that the wiki page you're citing states that individuals without an appendix were more likely to develop a recurrent C. diff infection. One could interpret that as they did on that site that somehow the appendix is repopulating the colon and making that individual more resistant to a repeat or "Recurrent" infection. However, I don't know of any studies that have directly tested that hypothesis.

In terms of whether or not the gut flora - aka "gut microbiota" - are significantly different in the appendix is possible, but I don't think anyone has identified that just yet. One of the problems with this would be that we don't routinely remove appendices unless they are infected/inflamed which intrinsically would alter the microbiome locally.

So, is it possible that the microbiome could be significantly different in the appendix itself compared to the cecum just a few centimeters away? The answer would be - potentially, but no one has been able to address that question to date.

human biology - Why is rabies incurable?

This is because rabies is a viral infection of nervous tissue that propagates through peripheral nerves into the brain and causes brain tissue inflammation (encephalitis).

As long as the virus is in the brain there is no way to get rid of it. The main trade-off here is that everything that would kill the virus will be as (or even more) aggressive against the brain tissue, and impairment of the latter will lead to really heavy deficits in vital functions like breathing and thermoregulation.

The first manifestations of rabies are those due to brain damage. This means, the virus is already there and the brain is already fatally damaged.

transcription - determining genome-wide exogenous binding of pathogens to host genome?

I've read this paper where they specifically modify a region in the rice genome to ablate the binding site of a pathogen, Xanthomonas oryzae, and disrupt the hijacking of a gene network in the rice genome to the advantage of the pathogen.

It's an interesting concept but left me wondering how one would determine de novo the binding of these pathogen effectors into the host if one didn't know them. What would be the steps to determine where are the pathogen's TFs binding the host genome?

Edit: there is the possibility of not knowing which proteins bind to the host genome, so what would be the way of developing the antibodies in this case?

Reference:

What factors are known to affect evolution?

What is evolution?

The first step is to remind ourself of the definition of the term "evolution". Evolution is most often defined as "any change in allele frequency in a population".

Forces that drive evolution

Categorizing the processes that affect allele frequencies might be subject to issues of semantics. Without going into the details, we generally recognize 4 forces that drives evolution

1. Natural selection
   
   
   • Natural selection refers to the deterministic change in allele frequency due to a differential in fitness among different genotypes.
   
   


2. Genetic Drift
   
   
   • Genetic Drift refers to the stochastic sampling process of individuals
   
   


3. Mutations
   
   
   • A mutation refers to any spontaneous change (substitution, indel, chromosome duplication, etc...) in an individual's genotype.
   
   


4. Gene flow (aka. migration)
   
   
   • Gene flow refers to the transfer (migration) of DNA sequences among populations.
   
   

---

KennyPeanuts's answer, random mating and hardy-weinberg equilibrium

In his answer, @KennyPeanuts also talk about random mating. Random mating refers to the condition where the probability of two individuals to mate depends only on their respective fitness. Many people phrase random mating as absence of mate choice but it actually refers to the absence of variation for mate choice in the population.

Hardy-Weinberg states that under the above four conditions and random mating, then the frequency of the genotype that has the allele $i$ derived from the mother and the allele $j$ derived from the father, where $x_i$ and $x_j$ are the frequency of these alleles is $cdot x_i cdot x_j$. This means that for a bi-allelic locus, the allele frequency of the genotypes AA, AB, BA and BB are $x^2$, $x(1-x)$, $x(1-x)$ and $(1-x)^2$, respectively where $x$ is the frequency of the allele A. For the heterozygotes (AB and BA), we often care little which of the two allele is inherited by the mother and which is inherited by the father (assuming there are genders) and we therefore call AB both AB and BA genotypes (which can eventually be confusing). As such, the frequency of the AB genotype is $2 x(1-x)$.

The condition of random mating ensure that there is no deviation of genotype frequencies from the Hardy-Weinberg's expectations and it ensure that there is no change in genotype frequencies from the first to the second generation considered (after one generation, the equilibrium genotype frequency is immediately reached). Random mating is therefore not a condition for evolution to not occur.

microbiology - Vigorous shaking for HFR interrupted mating

I remember doing this experiment many years ago in an undergraduate practical where we used vigorous vortexing of culture samples in glass tubes to achieve interruption and separation.

According to Griffiths AJF, Gelbart WM, Miller JH, et al. Modern Genetic Analysis. Bacterial Conjugation:

... sampling is accomplished by using a kitchen blender to separate
the joined cells, resulting in interrupted conjugation.

homework - Genetic carrier Pedigree of Recessive Traits

Let's take Question A:

Both fathers have siblings with red ears, and red ears are an autosomal recessive trait. The grandparents did not have red ears, we know then that they were carriers of the recessive allele. Each grandparent was Nr (for Normal and red alleles respectively).

The fathers have normal ears, so they could be NN (probability 0.33) or Nr (probability 0.67).

The answer then is that each father independently has a 2/3 chance (0.67 probability) of carrying the red ears gene.

The male's mother, being RR, makes the male a carrier only if the male's father is himself a carrier. To put it mathematically, he has a 1/2 chance of being a carrier with 2/3 probability that he was a carrier to begin with. (2/3)*(1/2) = 1/3.

Therefore the male can be carrier with 1/3 chance.

Now Question B:

Short answer:

Since the male and female have symmetrical pedigrees, and we just solved the chance of the male being a carrier with 1/3 chance, then the chance of them having a child with red ears is (1/3)*(1/3) = 1/9.

Long answer:

The male's and female's fathers may each be heterozygous or homozygous normal, given the information, with both mothers being homozygous normal (NN).

If Father A is NN (crossed with Mother A, also NN), there is a 0 probability of passing on the red ears allele.

If Father A is Nr, then Person A has a 0.5 probability of carrying the red ear allele.

Person A can now be NN (with 0.25 probability) or Nr (with 0.5 probability).

Likewise with Person B.

Now lets consider all 4 combinations of genotypes that Person A and B can have when they have a child. I will write a table with one Person on each side of the table, with the probability of their genotype written in brackets beside. Each intersection will represent the frequency of carrying the red ears allele.

                Person B
             (1/3)     (2/3)
Person A      NN        Nr
(1/3) NN      0/4       2/4
(2/3) Nr      2/4       2/4

Carrying the red ear gene: (A more interesting example)
There are a total of 6 outcomes in which the child carries the red ears allele, but these must be weighted by the probability that each parent has the associated genotype.

Probability of carrying 'r' = [ (1/3)(1/3)(0/4) + (1/3)(2/3)(2/4) + (2/3)(1/3)(2/4) + (2/3)(2/3)(2/4) ] / [ (1/3)(1/3) + (1/3)(2/3) + (2/3)(1/3) + (2/3)(2/3) ]
= 0.25

Having a child with red ears:
Probability of having red ears = [ (1/3)(1/3)(0/4) + (1/3)(2/3)(0/4) + (2/3)(1/3)(0/4) + (2/3)(2/3)(1/4) ] / [ (1/3)(1/3) + (1/3)(2/3) + (2/3)(1/3) + (2/3)(2/3) ]
= 1/9 or about 0.11

It's left as an exercise to the student to derive the remaining probabilites.
Nr 4/9
NN 4/9
rr 1/9

genetics - Bicoid regulation of hunchback

I'm learning about development via the example of Drosophila embryogenesis. I understand that bicoid regulates hunchback, among other genes. My question whether the regulation is direct or indirect? In other words, does the level of bicoid directly govern the expression of hunchback, or are there steps in between?

What temperature should mammalian B-Cells be stored at outside of the incubator?

I'm working with murine B-cells. The general protocol is to keep cells on ice to keep them from dying but I've noticed that it makes these cells aggregate and precipitate out. I've heard suggestions that these cells should just be kept at room temperature. How would I be able to determine which conditions at which I should be keeping my cells?

I was trying to collect cells to prepare them for cell binding studies to antibodies and then assess the cells on a filterplate. Typically, you keep them on ice to pause the metabolic state as well as to prevent endocytosis of the bound molecules. However, that shift seems to cause settling. How do you troubleshoot the appropriate temperature to process cells and what are the usual scientific justification for making such decisions.

gel electrophoresis - How many agarose gel bands are typical for circularised DNA

I don't have hands on it, but I will not be surprised if supercoiled DNA migrates at different distances according to some inner topological conformation (i.e., more or less coiled AND/OR more or less nicked). Similarly, this picture highlights >8 conformations. What is run in the gel is circularized phage DNA with some degree of knotting due to the circularization (phage DNA is otherwise linear).

Figure from: Arsuaga et al. 2005, PNAS 102 (26) 9165-9169 (free on PubMed Central)

Another theoretical possibility is that two or more plasmids of different sizes were grown in the same maxiprep because of some contamination. But in this case, you will spot the problem also in the digestion, so it does not seem to be your case.

evolution - How did the huge dinosaurs cope with gravity and loads on bones, etc.?

Assuming that gravity was essentially the same (other answers to this question notwithstanding), very large dinosaurs were dealing with the same forces that they would today. There are two clades of dinosaurs in which gigantism evolved, Sauropoda (quadrupdeal sauropods) and Theropoda (including T. rex). Each "solved" the problem of large size in different (but also somewhat similar) ways.

The main reason why large size was not a problem was that, if posture changed to align the forces between the animal and the ground, the bones are compressed. Bone is very strong in compression.

Theropoda

Theropod essential operate as a see-saw, with a large muscular tail balancing a large head. As such, they did not likely use much active muscular force to balance. The analogy is a human standing. Just standing, you don't need much muscle force to balance.

Hutchinson and Garcia (2002) showed that, because of a lack of plausibly large leg musculature, T. rex could not run. For a range of postures, they estimated how much muscle would be required to balance the animal and found that running behavior was unlikely.

Hutchinson, J.R. and M. Garcia. 2002. Tyrannosaurus was not a fast runner. Nature 415:1018-1021.

Sauropoda

Sauropods show many similar adaptations as elephants, the largest extant land mammals. Their limbs were held upright (erect), which requires less energy for balance. Some sauropods had air-filled bones, which would also lighten the skeleton. Wilson and Carrano (1999) document the evolution of posture through sauropod evolution from a biomechanical perspective.

Wilson, J.A. and M.T. Carrano. 1999. Titanosaurs and the origin of “wide-gauge” trackways: a biomechanical and systematic perspective on sauropod locomotion. Paleobiology 25:252–267.

metabolism - What are the differences between white and brown adipose tissue?

Not sure what you are asking, except to add to the list?

Its worth mentioning that brown adipose tissue is the only organ in the human body whose primary purpose is to generate heat. We are warm blooded, but the body temperature is regulated by other organs generating heat while they do work (like muscles or I suppose the stomach, kidney, etc).

Brown Tissue is supposed to not a juvenile attribute - doesn't show up in adults to the same extent (usually being limited to neck and upper chest.

Brown Tissue is thought to be present in only critical areas of the body - even in infants. Its found in the inner body cavity around vital organs.

The color comes from the large number of mitochondria in the cells, which is where the heat is generated via uncoupling protein 1 (UCP1).

White adipose tissue is what we more commonly call fat tissue - its primary function is to store energy in the chemical form of long chain fats. Fat tissue as it grows can inhibit the function of insulin in the body, increasing insulin resistance.

You could almost call them 'good fat' and 'bad fat'

neuroscience - Do taller people have larger somatosensory cortices than short people?

I would say no.

Your question somehow implies that somatosensory cortex is equally distributed among the different sensory areas of the body, whereas in the reality this is the known "cortical homunculus" concept of how this distribution looks:

If you look at this representation you'll see that chest and legs take only about one third to one fourth of the cortex, thus the changes between short and tall people don't seem to have noticeable contribution to the overall cortex size.

If we just walk away from cortex size and discuss the amount of nerve cells in these areas I would say that these numbers should correlate with the number of peripherial receptor cells that provide input for them, and since there is no known changes in receptor density among different body sizes, even the amount of nerve cells seems to be more or less same.

What is a good Non Fluorescent DNA Dye?

Can anyone suggest a dye which specifically targets DNA, but is not fluorescent?

(We plan to mark DNA before observing it with RAMAN-Spectroscopy. Because of the weak Signal even a low emission would disturb the reading)

Any tips would be much appreciated.

immunology - Organ cloning - possible to make a non-antigenic organ?

From a J. Neil Schulman article on Organ Cloning:

Cannibalizing organs from other people also entails the risk of
rejection because of incompatibilities, not only for tissue-typing but
also for gross anatomical mismatches. Cloning organs [...] has the
potential of taking a human being's own genetic material and growing
perfect replacement organs which are fully compatible with their
genetic makeup.

The reason for using a person's own DNA, is obviously to avoid an allergic reaction to the antigen markers on a strange organ. But in the blood typing system, there exist types which don't have any trouble-making antigens (such as type O-negative, called a "Universal Donor").

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Would it be possible to grow an analogous "universal" internal organ, which didn't have any antigens indicating it was a foreign body? (By editing a DNA sample to remove certain expressed proteins, then growing it into an organ.)

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I realize this is technically a science-fiction question :) But - A: it may not be in the near future, and B: I can rephrase it to remove the SF component:

In the blood-typing system, the AB antigens don't appear necessary for proper functioning of red blood cells - are the antigens on a person's own cells, actually needed for any essential functions, or could they be removed, without affecting that person's health?

How do you break up cell clumps when passaging?

In some protocols for setting up primary cultures (for example from mouse bone marrow or rat endometrium), there is a step that requires pushing cell suspension through a large needle to get rid of clumps. Of course, this carries a high risk of damaging the cells, so sometimes collagenase is used instead.

It might also be helpful to wash your cells with PBS without ions before passaging and use tripsin with EDTA. That should get rid of at least some calcium from the plate, so that action of adhesion proteins would be inhibited.

cellular respiration - What effect does ambient temperature have on the cardiovascular system?

I am sorry if this is not appropriate for this site, but I think it fits so I am asking it here:

I went for a run yesterday, and it was about -8⁰C. I was wearing a couple pairs of shorts, a t-shirt and a long sleeved shirt, which is not that much, so I went with the idea that I would warm up a lot while running. After running for a while, I found that I was tired and out of breath much earlier on then I would normally be. It raised the question:

How does decreasing the ambient temperature affect the cardiovascular system?

There were two things I could think of that might directly affect it. (1) The fact that my body's metabolism has to work to heat itself, and this takes energy. However I am not sure how much of a role this plays since running warms you up anyway. (2) The air being so cold, and entering the lungs, I imagine my body has to work much harder in some way to deal with it. What exactly happens here, and how is the process different from breathing at 25⁰C?

Also, how much faster would we expect a runner to be able to finish a 10km run when it is 25⁰C outside versus -5⁰C outside?

human biology - Is there such thing as a generic "metabolic type"?

A while ago I picked up a book called "Eat right for your metabolic type". The book discussed the effects of the endocrine system on the overall shape and the speed of metabolism.

The book outlined few major metabolic types:

• Adrenal


• Thyroid (hypo/hyper)


• Muscular

The book proceeded to discuss the overall shape of the body, including shape of fingers to help identify the metabolic type and give suggestions on which foods to avoid for a particular type.

The book then gave examples of effects of foods on metabolic types - for example an Adrenal type who eats too much sugar would crash and compensate for this by sleeping.

Since I can no longer find that book in print, I'm interested if there's really such a thing as a metabolic type that can be easily identified ? Are there indeed some foods that people should avoid eating?

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

I've read that many plants have some sort of circadian rhythm where they perform a certain action on a cycle of about 24 hours, like the mimosa plant opening and closing its leaves. Obviously this is done in order to synchronize with the sun, but many such plants continue to perform these actions even when left it constant darkness or light. It follows that this is the result of some sort of biochemical pathway, but whatever internal 'clock' is used is also independent of temperature.

How can these plants perform certain actions every 24 hours at different temperatures when the chemical reactions most biochemical pathways use are temperature dependent?

It would seem like the length of the cycle should vary widely according to the temperature as it affected the reactions involved, but instead it stays at a very constant length very close to 24 hours. How can this be?

(This is in my textbook, but unfortunately the book doesn't cite any studies. I would greatly appreciate it if anyone could help me find one to link to so I can improve the question. :)

biochemistry - What are the limitations to current nucleotide sequencing technologies?

It seems like you answered your own question,the signal from a few molecules running through an enzyme or a polymerase tend to fall out of synch after a few hundred bases. If an enzyme for sequencing was more rigorously in time step that could help for instance. The machines read traces in four channels with nice bumps for each base. See this article for a nice example. You can see that if there are too many of the same base consecutively it gets hard to tell how many bases there are there. Over time all four traces will start to smear out and you cant tell Adam from Thelma if you take my meaning.

But there are other bottlenecks.

The sequencers currently put out such a high volume of data that the analysis of the meaning of the output data can't be analyzed fast enough. This follows the trend in biotech over the past 12 years or so - more sequence data, micro array data, more mutation data, more genomes than people who can actually use it to understand the biology. There is a bit of an analysis bottleneck now.

So some of these sequencers have greater read lengths, which can make it easier to assemble a sequence. These sequencers generally cost more. For instance if you have a library to sequence a little fungal or algal genome - you will get the answer back in a day or less now. In the form of 1 Tb of reads maybe 50 to 200 bp long. It might take quite a bit of time to put that together into a novel genome sequence, still more to find the genes, build the gene networks from a template of pathways etc. Just imagine a thousand sequencers pumping out day and night and you get the picture I'm trying to paint here.

About cost. Ion Torrent and the new oxford nanopore sequencers are really cheap - $50k to perhaps $900 for Oxford Nanopore's USB sequencer. Most other systems cost hundreds of thousands of dollars. Ion torrent and Nanopore have more disposables - you throw away a chip or even the entire sequencer - at a cost of hundreds of dollars a sample.