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What are the highest and lowest possible pH, paCO2 and HCO3 in the blood of living human?
What are the highest and lowest possible values of pH, $paCO_2$, and $HCO_3$ in the (arterial or venous) blood of a living human being?

You should look for the standard reference ranges for arterial blood gases, which will give you your answer

These give the normal values in normal people, not the possible in any living human body.

How would we know the numbers for any possible living body? The best you could rely on would be individual data points from someone whose blood happened to be checked for those quantities (i.e. highest and lowest recorded concentrations). Not a controlled experiment. And no way to tell if that's the highest possible.

Perhaps a more reasonable thing to find is the variance or standard deviation of the values of these quantities in normal patients.

Thank you @A.Kennard. I understand that. My question would be better rephrased as "what are the ever known highest and lowest values". This article mentions a range of pH between 6.8 and 7.8. A simple example why one may need an answer to this question is when a spreadsheet should reject the entry of "impossible" blood gas values in a living human.

Practically, in order for this to be answerable you'll need to comment on whether you consider a pulseless person during resuscitation efforts to be "alive." It's not uncommon to see a pH ~6.8 range drawn during CPR, but that's a different situation than normal "life."

Good point @Susan. I think it is sensible to include all   cases with successful resuscitation.

As discussed in the comments, there are theoretical and practical problems with arriving at such data. There have been controlled experiments using canines, but you have specifically stated your interest in humans. I offer a summary of a few observational studies that may be helpful.
In this study, 117 patients in intensive care units (who are likely to have the most abnormal blood gas parameters among the living) were monitored with continuous optode-based blood gas analysis system. Of the 1,341 measurements, the ranges were:1,2

pH: 7.14 to 7.64
PaCO2: 19 torr  - 98 torr
PaO2: 38 torr – 413 torr3

Another study included a total of 487 samples were obtained in ICU patients with severe respiratory failure and found the following ranges:

pH 7.23 – 7.55
PaCO2: 19 torr – 83 torr
PaO2 30 torr – 522 torr3

These are reasonable ranges for pseudo-stable ICU patients.
Contrast that with normal ranges (+/- 2 standard deviations):

pH: 7.35 – 7.45
PaCO2: 35 torr – 45 torr

On the other hand, you’re interested in the most extreme recorded values, which are likely going to occur during cardiac arrest. According to this case report and literature review, the lowest published arterial pH of an adult who survived cardiac arrest with neurologic recovery is 6.33. The caveat here is that this was a near-drowning victim, and the associated hypothermia provides neurologic protection. The authors go on to report several others including their own patient with pH in the range of 6.5 - 6.6 with neurologic recovery in the absence of hypothermia.

Notes

1. Bicarbonate measurements on whole blood are calculated rather than measured and so not included in such studies. The more accurate test for this measurement is serum chemistry. The bounds are usually defined by the assay. It’s possible for a living human to have HCO3 less than assay (5 mmol/L in most labs by serum chemistry) or greater than assay (~50 mmol/L by most labs).

2. All data provided are arterial. Venous blood gases are of little clinical utility except as a cop-out for obtaining an arterial blood gas.

3. These upper values are intubated patients breathing 100% FiO2. (The lower values are those about to be intubated breathing 100% FiO2....) The theoretical maximum PaO2 on room air is derived from the alveolar gas equation, ~100 torr.


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How does an embryo know where to grow limbs etc
For example you have a cell or already a bunch of cells. Those cell(s) divide and after several week you have a grown organism, for example a human with limbs, several different organs etc.
However, how do cells know where up, down, left, right etc. is?
I know left and right can be defined clearly in physics, for example by the Wu experiment verifying parity violation really occur.
However how do cells define those directions? What are the mechanisms for them to grow into a certain direction and tell other cells to attain a certain property?

See Human embryogenesis and Prenatal development on Wikipedia.

You may want to look into "Evolutionary - Developmental Biology", which deals with how an embryo develops into a grown organism.

Thus, by combinatorial specifying the identity of particular body
  regions, Hox genes determine where limbs and other body segments will
  grow in a developing embryo or larva. A paragon of a toolbox gene is
  Pax6/eyeless, which controls eye formation in all animals. It has been
  found to produce eyes in mice and Drosophila, even if mouse
  Pax6/eyeless was expressed in Drosophila.

In short, there's a sequence of gene activations that progressively map body into different regions. At first, there are just 3 regions (head, body, tail), then these get broken down into more and more finely defined sections.
You can think of these sections as GPS coordinates for the body. A gene combination at a certain point means that a limb will grow there.
You can look into "Sonic Hedgehog" gene, which allowed scientists to produce fuzzy growth on a fruit fly head.
The most interesting thing is that every living creature is mapped/built using the same toolbox of genes. The genes that define where a fruit fly wing is also define where a human arm is.

I wanted to add some helpful references. The 6th edition of the Gilbert Developmental Biology textbook is available on NCBI bookshelf. It's a bit old (2000), but much of the information is still relevant. You can search this textbook for specific terms but not browse.
There is also a collaborative science/fashion project between the Storey sisters, called Primitive Streak, which documents the early stages of human development.
For any given developmental process, there are a number of genes being activated and/or inactivated at that stage that determine how and when structures grow. Events can determine axes too; for instance, the dorsal/ventral axis of the embryo is determine by how the embryo blastocyst implants in the uterine wall. The left-right axis of the body is determined by the direction that fluid travels through a structure called the node, which is directed by hair-like structures called cilia. If nodal flow is backwards, your internal organs will switch sides, called situs inversus.
Back to the limb example... in tetrapods, or animals with 4 limbs, the area where limbs emerge is based on Hox gene expression, and whether or not it will become a forelimb (arms) or hindlimb (legs) is determined by either Tbx5 or Tbx4 gene expression, respectively.
The proximal-distal axis (proximal = towards the trunk; distal = away from the trunk) is determined by FGF gene expression, which induces formation of a structure called the apical ectodermal ridge (AER).
The anterior-posterior axis (head to tail) is determined by Sonic hedgehog gene expression in a region called the zone of polarizing activity (ZPA).
The dorsal-ventral axis (back-front; or back of the hand/arm - palm/inside of arm) is determined by the Wnt7a gene being expressed on the dorsal side.
The outgrowth of the limb is determined by Hox gene expression.
Digit (finger) formation is dependent on cell death between the fingers - hands start webbed, kind of like ducks, and then genes such as the BMPs cause cells between the fingers to die.
Terms to look up for more detail, since I don't have enough street cred here to post more than 2 links yet:

Snapshot Summary: The Tetrapod Limb (Gilbert book)
apical ectodermal ridge (AER)
zone of polarizing activity (ZPA)
situs inversus
Hox genes

That node-flow ciliar thing causing situs inversus is so amazing!

yea the hox genes are essential for the the specification of the location but the actual growing of the limbs are determined by a series of protein and other factors. proximal limb contains fgf(fibroblast growth factors) wnt(look up wnt pathway) and high levels of retonic acid where distal limbs shows high fgf's high wnt and little to none RA. the sonic the hedgehog gene talked about in the other answer has to to do with the anteriorizing(up)/posteriozing(down) of the autopod(digits, carpals). as to dorsal ventral wnt would be the culprit

Can you please work on your answer and also add some references to it? At the moment it's hard to understand without basic knowledge of the field.

I concur with @Chris: with some elaboration here and there and the necessary citations this could be a great answer

why would i have to cite topics that i have learned through school, it is a topic that i am familiar with as far as elaboration sure what exactly would you recommend i elaborate on.

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Hela live cell confocal laser scanning - reccommendations for good fluorophore that will show good movement
I've been doing a lot of live cell imaging lately mostly using hela cells expressing some EYFP based chimeric proteins. I'm building a video library for an art student here at the university who is going to create an exposition using the videos. I need a lot of content to make this sucessful and rather than requiring much scientific merit these videos need to be "busy" and look good. I'm looking for suggestions as to any dyes or fluorophores, drug treatments or conditions someone has worked with that will show significant movement/change of/in the cell, change in intracellular localization....doesn't matter it just needs to be a significant alteration for the cell.
I'm still really optimizing the system with the CO₂ etc.. and the longest aquisition i have taken is about 8 hours - after that either the cells are dead or the focal drift becomes too much of a problem. I am hoping to see some division once I can get the system recording long enough. Until then was looking for some suggestions as to other possibilities.
The various dyes I have been using include:  hoechst, dapi, mito-tracker, draq5, er-red, rox-red. also eyfp/tubulin, eyfy/mito, eyfp/golgi, draq5. These dyes aren't cheap and I'm only one i know doing this so I'm checking here.

I am not really sure, what you are asking for. You are looking for a fluophore that is nicely visible in confocal microscopy? Or do you want to tag a protein, which moves a lot? Or do you want to see whole moving cells?

Thank you Chris, I added a lot of needed info to the original question.

You could think about tagging proteins, which are imported and exported from the nucleus.

MitoSOX looks pretty cool. I've never used it myself, but I'm sure you could see some cool mitochondrial dynamics going on. Its a bit pricey though, but all dyes are going to be.
Even at only 8 hours you should probably be able to see a few divisions, which would be cool with hoechst.

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Inoculation vs. vaccination
Is there any actual difference between inoculation and vaccination or are these terms interchangeable?
In case the difference exists, would it be correct to say that inoculation is purposefully infecting a person with a pathogen in a controlled way, even when the person is already infected, to induce immunity while vaccination is administering dead or weakened pathogens to a healthy person as a preventive measure to check future infections?
Also, would it be correct to say that vaccination is an advanced form of inoculation?

Both are forms of immunisation.
Inoculation is exactly that. A live organism is introduced in a controlled way, so as to minimise the risk of infection, and is essentially the same process followed by many people in history.  It is inherently risky.
Vaccination is introducing a weakened version of the pathogen, so that the immune response is triggered and the body is prepared to fight the actual pathogen if necessary. This was pioneered by Edward Jenner, wherein he noticed that cowpox (related to smallpox) immunised the milkmaids against smallpox.

Cowpox (Vaccinia) is not "weakened smallpox" (Variola). They are entirely separate, but related, viruses.

@MattDMo True. It slipped my mind. Corrected the post.

Interestingly, the word vaccination is derived from vacca,  the Latin word for cow.

I'd maybe clarify. A vaccination isn't necessarily the whole pathogen, like the tetanus vaccine is the injection of a tetanus toxoid prepared from the toxin, so you build antibodies toward that (inactive) structure. Likewise, Gardasil is simply inactive viral proteins from HPV. Flu vaccine, however, is indeed attenuated flu strains. I would argue the fundamental difference is inoculation used live organisms where this isnt necessarily true of a vaccine in the modern sense.

@RoverEye That helped. However, I am still not sure if it would be correct to say that vaccination is an advanced form of inoculation.

In a general sense and as per the current scientific parlance, inoculation is used to mean introduction of a microbe to a system. This can refer to immunization procedures as well as for addition of a microbe to a culture medium in microbiological procedures.
Vaccination specifically refers to an immunization procedure which may involve attenuated pathogens, inactivated toxins (tetanus toxoid) or even specific proteins which are expressed in a lab microbe using recombinant DNA technology (as in case of hepatitis-B vaccine).
Etymology of both the terms, however, pertains to smallpox immunization. "Inoculation" is derived from Latin inoculatus, past participle of inoculare "graft in, implant a bud or eye of one plant into another [1]. In this procedure, the immunization was done by infecting an individual with a smallpox pustule from a patient (in a way grafting the pox).
"Vaccination" comes from the latin word vacca which means cow. This refers to the vaccine that Edward Jenner derived from cowpox.

This is much better answer alhough seems to be less liked. When we transfer microbes to a petri dish, it is called innoculation, although it has nothing to do with immunuty.

If you got your flu shots you could say that you got your vaccination, your immunization or your inoculation
and you would be correct with all three words.  However the words do have different yet overlapping meanings.
Vaccination is the act of administering a vaccine. Although the word "vaccine" started with Jenner's cow pox it was expanded by Pasteur to mean all inoculations intended to perform immunity. The vaccine is a pathogen or toxin rendered survivable either by altering it or,  most commonly, by using minimal amounts.
Immunization is any part of the process of becoming immune, but particularly the part taking place within your body as a response to a vaccine. You could gain immunity without a vaccine, and you could get a vaccination without gaining immunity.
Inoculation is a wider term for any transfer of tiny amounts of a material to a living organism, but the word has expanded beyond biology and is used in varied ways. An inoculation does not need to be for the purpose of immunization although immunizing is the most common usage. Intentionally infecting a lab mouse with a disease is inoculation but not intended to immunize. The original use of the word was to graft seeds (also called "eyes" thus the "ocula" etymology) which has nothing to do with immunity.
In some medical circles, inoculation is skin administered,  while vaccine is oral or both oral and skin, but there is no such concise definition by universal agreement.

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Stress regulation in mosquitoes
I was just trying to understand how in insects, specifically mosquitoes does the process of emergency responding, stress regulation take place? Or in simpler words how are flight, fright and fight situations handled?
I understand that insects have a ganglion system and they do, suitably, respond to external stimuli, but I would like to know if there is any chemical coordination that works specifically in emergency situations just as adrenalin and nor-adrenalin do in our case.

see this

Does this mean that besides inducing long term effects, as suggested in the article, dopamine also induces the same temporary effects (like flying with greater speed and trying to escape when in danger) in insects as adrenalin does in our case?


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Why is cold water more refreshing than warm water?
Obviously, the temperature of water does not affect its chemical composition. At least not in the ranges we are likely to drink it in. Yet it is clearly far more pleasant and refreshing to drink cool water than it is to drink tepid or warm water.
Is there actually any difference to the organism or is this just a matter of perception? Is cool water somehow more efficient at rehydrating a cell? In any case, surely by the time water reaches individual cells it will have warmed up to body temperature.
So, what, if any, is the difference between drinking cool and warm water in terms of its effect on the human (or other animal) body?
Extra bonus for explaining why the taste of water changes when it is cold.

However, on a hot day an hot drink will make you less thirsty than a cold one.

Care to elaborate @nico? Are you saying that having a drink will make you thirsty?

@terdon: no, I am saying that a hot drink will make you feel less thirsty on the long run. For instance we always bring hot tea when hiking in summer, even if it's quite hot. It's a matter of temperature difference, like having a hot shower in summer, you'll feel way more refreshed then after a cold one.

@terdon: If your drink is cold, your body has to heat the drink up to body temperature. This consumes energy, which leads to a higher body temperature, which leads to sweating. The net fluid balance can probably be negative. :-) That's why desert natives drink hot sweet tea.

@MartinSchröder, yes I understood from Nico's answer. That makes sense. It does not, however, answer the question. Indeed it contradicts it :). Why do we feel more refreshed when drinking cold water? Is it just a question of perception restricted to those surfaces that come directly into contact with the water (mouth, throat etc) or is there a deeper physiological reason as well?

@nico, I understand what you mean and the theory behind it and you are of course quite right. Nevertheless, I have never felt a craving for room temperature water. I often have for cold water. Why not? If drinking warm water makes feel cooler because of the temperature differential with our environment, why then do we not crave hot water on a summer day? Most cravings have a sound biological basis.

@MartinSchröder  Doubtful.  The cold material will absorb energy that's already present.  Your body doesn't have to do any extra work for this to happen.  It might be plausible that a cold drink will increase heat stress on the body in more subtle ways though.

Cultural perception, I know cultures that do not drink anything that hasn't came out of heater

@MartinSchröder, sorry, but you are wrong, it is quite the opposite, actually. Many cultures drink warm or hot beverages in hot days, because the extra heat from the drink increases your core body temperature, which in turn causes sweating. Guess what sweating is designed for. :)

Short answer: Cold is pleasant only when your are not already freezing and cold might satiate thirst better because it acts as enhancer of the "water intake flow meter".

Is cold water more tasty than warm water? No, it is actually the reverse as detailed in my footnote.
Cold is pleasant when your body is over-heating and definitely not if you live naked in the North Pole. Over-heating means sweating which means you loose water and therefore feel thirsty faster. Yet drinking cold water will not rehydrate the body more than warm water and drinking water has only a very small impact on the body temperature. So why do we like it?
A study was actually conducted on the subject and answers most of your questions. Here the reference.
The temperature of the body will indeed not change.

cold stimuli applied to the mouth
  (internal surface of the body) do not appear to impact on body
  temperature and are not reported to cause any reflex shivering
  or skin vasoconstriction that influence body temperature.

As you pointed out, the temperature of the ingested water will not affect the overall hydration of the body as cells are rehydrated mostly via the blood  stream and the blood temperature will not be affected. Someone could argue that, at identical volumes, cold water (above 4C) contains more molecules (i.e. is denser) than warm water but this difference is likely very slim.
In this paper they also define "thirst".

Thirst is a homeostatic mechanism that regulates blood osmolarity
  by initiating water intake when blood osmolarity increases

The problem is that it takes some time before the water reaches the blood stream, and therefore you need a feedback mechanism that tells you to stop drinking independently of the blood's osmolarity. This is where cold might play a role.

The cold stimulus to the mouth from ingestion of water may act
  as a satiety signal to meter water intake and prevent excessive
  ingestion of water

The picture would then be the following

In essence, a cold sensation is pleasant in warm weather, both on the skin and in the mouth, and it apparently helps in reducing thirst by being some kind of an  enhancer of the "water intake flow meter".

Footnote
Reading the comments I just want to clarify some points.
The 5 basic tastes (sweet, salty, bitter, sour and umami) are very distinct from taste sensations (pungency, smoothness, cooling to name a few). The main difference is that taste and "sensation" signals use completely different paths to reach the brain - namely, the facial and glossopharyngeal nerves for the former and the trigeminal nerve for the latter.
Is the temperature affecting basic taste perceptions? The answer is yes. How this happens is quite simple if you understand the fundamental concepts of molecular taste perception. Essentially the temperature affects the response of the receptor TRPM5 which is the main player in depolarizing taste receptor cells in the papillae. To put it simply, higher temperatures provoke a greater perception for taste, and this is not only in term of perceived taste but really modifies the amplitude of the response at the molecular level. As an example this is why ice cream does not taste sweet when frozen but only after it melted in the mouth or on the tongue.

you are absolutely correct. It is the same if you live naked on the North Pole - you will find warm water more pleasant than cold. :)
However please do note that stimuli to the mouth may not affect body temperature but stimuli to the stomach (i.e. almost core of the body) WILL affect body temperature.

Here's an armchair evolutionary explanation. In nature, running water tends to be cold whereas tepid water tends to be lukewarm. And for reasons having nothing to do with temperature, running water tends to contain less harmful bacteria. Therefore, our ancestors who preferred to drink cold water over lukewarm had an evolutionary advantage over those who preferred the opposite.

The organism as a thermodynamic machine does need a coolant. The more difference in temperature the more efficiently thermal machine can use energy. Also in all processes in organism it is produced excessive thermal energy, which should be removed.
Thus colder water and air are pleasant.


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How can I compare rates of evolution for two sets of proteins?
I have a list of candidate proteins as the result of my analysis. I am now trying to find various characteristics that they have in common. One of the things I would like to check is if my candidates are evolving faster or slower than the rest of the proteins in my dataset.
Now, I know how to do this manually by building multi species alignments for each of my proteins and calculating ka/ks ratios for each set of alignments. This, however, is not a trivial process and I really do not want to do this manually for my ~1500 candidates.
Can anyone suggest a tool that will take two lists of proteins and return an estimate of evolutionary rate for each one?
I am primarily interested in human but also mouse, yeast, fly and worm. Ideally, I would need a tool that can take a list of proteins, assign them to an orthologous group and return a value representing the rate of evolution for that group.

I think you'll have to build the MSA regardless of the strategy that you use. You could try faster tree-based methods.

are you working on species that have already been sequenced (for which you might info in databases), or do you have novel sequencing data?

@MichaelKuhn definately sequenced. I am working on human primarily. There should be a database around that can give me this information right?

The eggNOG database also contains alignments for protein families, but these are on the amino acid level. So you would still somehow have to go back to a nucleotide alignment, which I'm not quite sure how to do. :-/ Might be a good question to ask on BioStar with its larger bioinf audience.

@MichaelKuhn, protein data is fine, I just realized my original question was not clear enough and have updated it. I said genes in the original because I thought it would be easier to find a relevant tool. My bad.

@terdon: if you want to compute ka/ks ratios, you need to look at the nucleotide sequences, no?

@MichaelKuhn, yup, that's why I had said genes in my original question. Since you suggested a protein database, i wanted to make it clear that protein data is fine. It does not necessarily need to be ka/ks, any metric of evolutionary change will do. There could, for example, be a database out here that has calculated homology scores for protein families, that would be fine for me.

If protein alignments are sufficient for you, I suggest that you look at the eggNOG database. There you can download mappings from proteins to protein families, and for the families you can also download pre-computed multiple (protein) sequence alignments.
I guess depending on the type of analysis you want to do you would have to trim the alignments to get only homologous regions (for example, see this recent paper[1]).

Wu M, Chatterji S, Eisen JA. 2012. Accounting For Alignment Uncertainty in Phylogenomics. PLoS ONE 7(1): e30288, doi:10.1371/journal.pone.0030288.


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Identify this insect - beetle (?)
Please help identify this insect - genus, species and anything interesting about it. (Species of beetle?)
Old link - please vote to un-delete it https://biology.stackexchange.com/questions/64042/can-you-identify-these-insects?

That's a red rhinoceros beetle. Although, "red rhinoceros beetle" is not the correct name. I'm simply observing that it is red, and that it is a rhinoceros beetle. So, according to wikipedia: suborder Dynastinae. Do an image search for "red rhinoceros beetle" and you'll stuff that looks quite similar. Searching google for that specific image show's that it is a 3d printed model. The list of bugs in list of 3d printed models includes something called "Rozenkever". Which is remarkably similar to "Red Beetle" in German.
Golofa porteri perhaps

Is there any way we can get this down to the species identification? the missing link from above, long story short, the artist sent these files to me without proper naming for an entomology app I'm developing. https://biology.stackexchange.com/questions/64042/can-you-identify-these-insects?

golofa porteri seems likely then.

@Charles Translation makes no sense at all. Rozenkever is Dutch, not German and means "rose beetle", not "red beetle". Your link shows an image, with the name "Meikever", meaning "May beetle". However, this is not a meikever Melolontha melolontha.


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Alternative protocol for evaluating RNA integrity, using a bleach gel
I hate having to run MOPS PFA agarose gels with DEPC and everything just to verify my RNA integrity. I came across an alternative protocol that uses ordinary household bleach in the gel to inhibit RNases and was wondering if anyone here has tried it and what the success was like? Or any helpful insight is welcomed also.
here's a protocol I put together based on what was done in that paper.
For a 50ml gel
IN a RNase-free 250ml Erlenmeyer flask
Mix:
5 mls of 10x TAE that I gave you
42.5mls ultra purified water
0.5g of the agarose I gave you
2.5mls of household bleach (6% sodium hypochlorite, use clorox)
Incubate 5-10 minutes.
Microwave for the minim amount of time on the lowest heat setting that’s needed to completely solubilize the agarose, you don’t want evaporation adjusting your solutions volume
Check your volume with a sterile 50ml serological pipette, if you have lost more than 5% volume I would redo, using less heat.
Add 3µl 10mg/ml ethidium bromide, swirl to mix.
Pour in casting frame, allow to cool, submerge in 1xTAE
Combine 1µg of your total RNA, 2µg RNase free 6x loading dye and q.s to 12µl in RNAse feree water, you can use the water from the qiagen kit for this.
Run at 100V, about 30 min, get  good scanned image of it and use Photoshop to assess the band intensity. The 28S should be twice the brightness  of the 18S in a sample with little to no fragmentation.

I also hated the denaturing agarose RNA gels, but I switched to native gels using Faster Better Media's Lithium Boric Acid buffer at 0.5X concentration, my gels look much better, though I get 2 bands in each lane because it's not denatured. How are you using the gels to check for RNA integrity? If I see a band at the right size I've been calling it good enough and moving on. How precise does a gel need to be?

Looking at the small and large subunits. The large one should be 2x as bright in an unfragmented sample of total RNA. The gel needs to not exhibit any exonuclease activity, not much more than that needed

Maybe that's why I've been able to get away with my gels, I'm making RNA through In Vitro Transcription, you must be isolating it from cells.

You are correct!

What is your ultimate objective? What do you need RNA for? The rigor of the test depends on that. For usual RT-PCR I heat RNA with the 2x loading buffer that contains formamide and run it in normal agarose gel with TAE/TBE at 120V.

Testing RNA integrity for Microarray

Why not just run the RNA on an Agilent Bioanalyzer nanofluidics machine (or something similar)? It's fairly cheap and quick, and I'm sure a lot of core services offer it now. Of course, I am making certain assumptions about funding and technology availability here...

@Cantona'sCollar that's certainly the gold standard but I don't see how it's cheap those chips have a very supposed short shelf life for the bulk you have to buy it in. Also any fluids machine had the potential for disastrous cost.

I don't think bleach can denature RNA. Bleach is an oxidizing agent and it will damage the RNA. Also, the protocol mentions addition of hypochlorite before heating which I think is illogical because heat will decompose it.
For testing RNA integrity you need not make a denaturing gel. What you can instead do is to heat the RNA with the 2xRNA loading buffer that contains 95% formamide and run it in a normal TBE/TAE agarose gel. Heating with formamide denatures the RNA permanently.
Denaturing gels are only essential for Northern Blots.
You can also run your sample in a bioanalyzer to check for RNA integrity and this is always recommended for RNAseq experiments (even for microarrays but I cannot comment much on that because I have never done it and am not aware of the usual practice).

I'm telling you I didn't believe it either but it works quite well. Don't add it to the agarose before microwaving and use 6%. Also the rna will be denatured already from most any purification procedure especially the standard ones that use guanidine. works good for a teaching lab (cheap and easier) and I think this protocol focus's on RNAse removal or inactivation. +1 for the heating recc but I'm weary to heat rna unless it can be flash heated. I may Ty this next time however.

The bleach is not to denature the RNA but to destroy and RNase that might be lurking in your agarose or buffer - heat doesn't kill RNase easily so the boiling doesn't help.  Then just run a normal agarose gel (TAE/TBE or borate) and look at the relative intensities of the LSU and SSU RNA.  The bleach gels work very well for me and I've got very good RNA-Seq results out of RNA analysed this way (I don't have access to a bioanalyser).

Please add some references to your answer.


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What is reductive deamination?
This presentation on anaerobic digestion mentions (pg. 19) two major pathways for the digestion of amino acids: Stickland reaction and reductive deamination. While I find some info on the former, what is reductive deamination? What is a representative reaction and what partners are needed?

Amine is already a reduced state and therefore the reduction part is not of the amine. In chemical process of reductive deamination the arylamine is first converted to an intermediate, usually by sulphonylation of the amine nitrogen [ref], which in turn is reduced.
$ArNH_2 + CH_3SO_2Cl \ \ \  _{\overrightarrow{pyridine}}\ \ \  ArNHSO_2CH_3 \ \ \  _{\overrightarrow{NaH+NH_2Cl}}  \ \ \ ArH$
Biochemical anaerobic reductive deamination is reported but not for amino acids (I couldn't find any). It is reported for anilines which probably proceeds via a carboxyl intermediate [1, 2].
This article says that the non-classical amino acid $ornithine\ \ $ is converted to $\delta-aminovaleric acid\ \ $ via a $\ proline\ $ intermediate but no mechanism is described.
I don't think this answers your question completely; the best solution would be to ask the author of the presentation for the exact reference.


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Are There Exceptions to Animal Cells not Having Cell Walls?
In the January Issue of SciAm (discussing Haemophilia):

When damage occurs to blood vessels, exposure of the blood to collagen in the cell walls and material released by the cells triggers the activation of clotting factors.

I read the original in print, but it is available online here.
This seems to imply that animal cells (in this example, those of humans) have cell walls.  I sometimes see similar implications in other resources.  However, in elementary biology, one is taught that animal cells never have cell walls.
Therefore, my question: Are references to animal cell cell walls (such as the above, for human animal cells) simple mistakes--or are they exceptions to a generalization?

This was just a badly worded statement; the better wording should have been: "exposure of the blood to collagen in the blood vessel walls and material released by the cells...". They clearly mean damaged endothelium.

Can you also post a link to this article. If they have used this erroneous terminology then we should write to the editor.

@WYSIWYG see edit.  When I read the quote, I immediately suspected an error.  I do permit SciAm and the author the occasional typo, though, since they're obviously high-quality otherwise.

@imallett It is for everyone's good to report mistakes. I once found ~100 articles that had mistakenly called TCA-Cycle as Trichloroacetic acid cycle instead of tricarboxylic acid cycle. I mailed the editors.

Humans, as well as the rest of the metazoans (i.e. animals), absolutely do not have cell walls. What humans do have is extracellular matrix (ECM), which is the sort of fibrous, sort of gel-like material in which cells in many of the tissues are embedded. Collagen is a major component of ECM.
From the old copy of Alberts that is hosted on the NCBI website:

Tissues are not made up solely of cells. A substantial part of their volume is extracellular space, which is largely filled by an intricate network of macromolecules constituting the extracellular matrix (Figure 19-33). This matrix is composed of a variety of proteins and polysaccharides that are secreted locally and assembled into an organized meshwork in close association with the surface of the cell that produced them...
Two main classes of extracellular macromolecules make up the matrix: (1) polysaccharide chains of the class called glycosaminoglycans (GAGs), which are usually found covalently linked to protein in the form of proteoglycans, and (2) fibrous proteins, including collagen, elastin, fibronectin, and laminin, which have both structural and adhesive functions.

@anongoodnurse yeah, I figured it was something like that too. I just wanted to make super clear to the OP that, at least in terms of nomenclature, animals definitely do not have cell walls.

@anongoodnurse which got me thinking about whether cell wall vs ECM is a difference without a distinction, but I cut that part out of my answer as it didn't really add anything to it. I might put that up myself as a separate question once I've looked into it a bit.

authors: Лейля Галиханова

authors: آرسس یوسف وند


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Alternatives to commercially available flow cytometric analysis software for use in cell cycle analysis?
I know that many labs use either FlowJo or FCS Express to plot and analyze their flow data. These seem like the two standards for most labs (and I have used FlowJo before). However, my current funding does not cover software licenses and I'm not keen to pay $500-2000 out-of-pocket for a license.
Are there alternatives that approach the quality of FlowJo/FCS Express but are open-source/free with an academic license? I'm looking for an approach which will allow me to fit G1/S/G2-M curves to my data.

Meta-question on Meta: http://meta.biology.stackexchange.com/questions/743/is-my-flow-cytometry-software-question-on-topic

Excel would do perfectly fine :)
there are several plotting tools- R and Matlab have good plot tools. Get the text file out and plot anywhere you like. $500 is too much for that; just ridiculous. What you must know is how to analyze and what different parameters are— that you in any case must; you cant depend on a black box.

Sounds like you should look into Bioconductor's flow suite.  I actually really like doing flow analysis in R, but it takes some practice and getting used to.
The biggest problem you will run into (in my opinion) is gating.  It's not easy to setup a GUI point and click gate.  Rather, you'll be more forced and inclined to set limits with algorithms or hard value limits.
I found .Net Bio to be fun, but not nearly as powerful as Bioconductor.
Meta note, couldn't think of a less opinion oriented way to answer other than just to say "It can be done in R utilizing Bioconductor."  If this is still off, I'm fine with deletion.

I created a free flow cytometry tool that works on any device https://www.redmatterapp.com/
The benefits of this tool is that it works on any device and analysis can be shared via a URL.

Hi Mark. Instead of simply posting a link to your tool, please provide some context and perhaps screen shots. Your answer should be able to stand on its own as being useful/informative without the need to visit other sites. A short one sentence answer with a link is likely to be deleted. Thanks.


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Identifying a possible pathogen of a succulent plant (Haworthiopsis attenuata) presenting with small brown scales
I have a succulent (most likely Haworthiopsis attenuata) that is currently not looking so hot. I recently noticed a number of small brown scales growing on multiple leaves (Eastern USA), that I think are some sort of pathogen:

They don't appear to follow any sort of pattern like the white marks on the leaves.
They are easily detachable with tweezers--I plucked two or three off as a test.

The best explanation I've found myself is that they're some sort of scale insects, but they seem really tiny. These are about the size of a Chia seed--only a few mm across. What are these, and as a bonus, how can I get rid of them? Photos are below:

I also would say that these are scale insects. You could try brushing them with slightly soapy water. Maybe you will get better help at https://gardening.stackexchange.com/

license: Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/


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How to Extract RNA and perform RT-qPCR from very few cells ( ~5,000)?
I am currently conducting an experiment which involves FACS-sorting a specific population of cells using an antibody of interest.
In order to validate the type of cells I have collected using this marker, I want to perform qPCR on their extracted RNA, using certain known cell-type marker primers.
My problem is that in each sorting session, I am only able to collect anywhere from 5,000 - 10,000 cells. Do you know if it is possible to perform RT-qPCR on such a small sample size?
I have collected the cells in 1mL of Trizol.
Would I maybe have to amplify the cDNA after reverse transcription in a general manner ( using random primers)?
Thanks for your help.

Wouldn't hurt to try. With the right primers and enough PCR cycles you should be able to amplify just about anything, maybe not perfectly though.

I think this should be possible, although you will not get very much cDNA, so you probably have only one shot. Besides this: Why are you using so much Trizol, I typically used 1ml for one well of a six well plate, which has ways more cells. When you precipitate, make sure your are using some co-precipitation agent as glycogen, so you won't lose sample. And you will probably not see the RNA pellet with so few cells.

Thanks Chris. How much Trizol would you recommend? I'm using 1mL because I sort into 1.5 Epp tubes and try to prevent the cells from sticking to the walls of the tube when they exit the sorter. Would amplifying all the cDNA after RT make sense? (Non-specific, just random primer amplification?)

Aren't you going to amplify the cDNA in your qPCR anyway? A pre-amplification would probably cause trouble with your data.

I wouldn't pre-amplify the DNA, as this can bring a lot of problems with biasing the ratios between the RNAs. Even if your cells stick to the wall of the tube, if you vortex the tube, the cells will be lysed. See how much Trizol is recommend for which cell number, if I remember correctly, there is a part in the manual. Besides not needing so much Trizol, you will also save money and produce less harmful waste.

1ml trizol is quite a lot. I use that much for ~1 million cells (a well of a 12-well plate- for HeLa. You generally need half of that). You can also try one of the RNA extraction kits. Add DNAse-I to remove any traces of DNA. Don't do a random primer amplification. Take a look here

You can consider use of single-cell RT-qPCR kits such as this one if you have the budget for them. (Note: I have not personally used this kit, just putting out an idea)

The Ambion® Single Cell-to-CT™ Kit contains a complete validated
  workflow for gene expression analysis for samples containing 1–10
  cells. Each kit contains reagents for sample preparation, reverse
  transcription, pre-amplification, and qPCR that have been optimized
  together in a simple workflow that can be completed in only 5 steps
  (see figure).

With good technique, you can reliably perform RT-PCR or RT-qPCR on a sample size of about 100 cells. Some of the comments are correct that you will need to compensate for the reduction of the initial cell input with additional PCR cycles. However, each magnitude in reduction of cells corresponds to about 3 extra PCR cycles (2^3 = 8 ~= 10), so your overall qPCR cycle number will not change significantly.
Since you are dealing with a small amount of cells, and isolating by TRIzol, the pellet could be difficult to see. I would suggest you use something like Glycogen (Glycoblue from Ambion) to co-precipitate the pellet into something more visible. You would add this prior to the Isopropanol to precipitate your nucleic acids.
Your use of extra TRIzol is not a problem unless you're lab is short of money. It will dilute your concentration of nucleic acid, which will make precipitation harder, but using glycogen will compensate for this. Using an amount less than the manual directed ratios can be problematic as it could result in incomplete lysis and denaturation of your cellular components.
As user137 recommends, Pre-amplification of the products is not a good idea as it will likely distort your final results. I'm not sure what gene expressions you're looking at, but it is always a good idea to include multiple housekeeping genes as baselines to calculate your relative expression numbers, provided you have enough RNA isolate to use as input.


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Are epigenetic modifications the most stable mechanisms for cell differentiation?
Wondering what the general take is on what are the molecular mechanisms that are mostly responsible for cell type differentiation stability; ie, for a cell's identity to actually become 'locked in' and progressively less susceptible to extracellular cues (for example, in heterotopic transplantation experiments such as Sue McConnell's).
I'm pretty convinced it really comes down to DNA methylation / chromatin remodelling / histone modifications in order to reach those truly stable, self-perpetuating, differentiated cell identities, but haven't been able to find a paper or review that actually discusses this. Your suggestions and opinions are welcome.
Also posted on reddit/labrats
Edit: I suppose another way to ask this is: Are Waddington's stable "attractor cell states" most likely or most easily explained by epigenetic modifications such as DNA methylation?

Epigenetic marks are reversible (you might be aware of induced pluripotent cells). Many animals can regenerate organs with high tissue complexity (such as a limb) and this involves de-differentiation in some species (Sandoval-Guzman et al., 2014). Even otherwise, cells can respond to extracellular/environmental cues to modify their epigenetic state (for e.g. EMT involved in wound healing). I would still consider epigenetic marks to be more "stable" than simple regulatory mechanisms.
Only way for a cell's identity to actually become 'locked in' is by genetic changes. The maturation of lymphocytes (especially the B-cells), would be a good example. These genetic changes happen via V(D)J recombination, class-switching recombination and somatic hypermutation.
L1 retrotransposons are also known to contribute to somatic heterogeneity in the brain neurons. However, the frequency of insertions is not very high (Evrony et al., 2012).


text:
Do mitochondria use ATP in order make another ATP from glucose?
Do mitochondria use ATP in order make another ATP from glucose, or does it use another source of energy to convert glucose to ATP?

During ATP production through the citric acid cycle, some ATP is used to transport necessary molecules for the reactions.

piruvate molecules are actively transported into the mitochondrial matrix [2]
two NADH equivalents from glycolisis are transported into the mitochondrion and consume two molar equivalents ATP [1]
protons leakage across the mitochondrial membrane leads to reduced ATP from NADH and FADH2 [1]

In theory, one molar equivalent of glucose can generate 38 equivalents of ATP. The actual number is only 25 [1].

References:

Wikipedia contributors, "Citric acid cycle," Wikipedia, The Free Encyclopedia, http://en.wikipedia.org/w/index.php?title=Citric_acid_cycle&oldid=610117265 (accessed June 26, 2014).
Wikipedia contributors, "Mitochondrion," Wikipedia, The Free Encyclopedia, http://en.wikipedia.org/w/index.php?title=Mitochondrion&oldid=614223751 (accessed June 26, 2014).


text:
How are memories stored in the brain?
I've been thinking about this for a while and I can't figure out how a memory can be stored in the brain.
I understand that important memories are favourably retained, such as a near death experience, the birth of your child or your wedding day. But I cannot comprehend how this can be stored biologically in the brain.

Firstly, I'd like to emphasize that the understanding of memories and learning as we know it is still in its very early stages. In fact, the mechanisms behind short-term memory are still under intense debate.
As for long-term memory, it is essentially caused by chemical changes in synapses between neurons in the brain. When you sense stimuli from your environment, action potentials follow a specific trail of afferent neurons towards the central nervous system, which then sends a message back through efferent neurons to initiate a response. The high frequency of these action potentials from the same stimuli of whichever sense allows for the strengthening of specific connections between neurons, which is called long-term potentiation. This phenomenon is one of the major components of synaptic plasticity, which also applies to the weakening of synaptic connections between neurons due to lack of transmission, as well as the change in the number of connections the neurons are receiving and making. Synaptic plasticity is also influenced by the change in quantities of neurotransmitters being expulsed into the synaptic cleft, and how cells react to these neurotransmitters, which is highly regulated by calcium levels. Ultimately, synaptic plasticity in accordance with learning is referred to as Hebbian theory.
We are still not exactly sure where memories are stored within the brain, but we do know that the hippocampus does play a big part. It definitely aids in the storing and structuring of memories in the form of these millions of synaptic changes. Finally, memory consolidation occurring in others regions of the brain over longer periods of time is likely, but still debated by some.


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Solvent Accessibility, the 20% cut-off method
I'm reading the papers linked below and all three of them mention a 20% cut-off for buried/exposed residues, by calculating a relative solvent accessibility (RSA) value.
I understand how the RSA is calculated, by dividing the calculated solvent accessibility against its total solvent accessibility values from paper 4 table 2.
RSA = calculated/total
e.g. if arginine is calculated to have a solvent accessibility of 55.43 and its total solvent accessibility is 241 then the RSA = 55.43/241 = 23%, so this arginine is considered exposed (see statement 1 below).
What leaves me confused is the definition or lack of the 20% method for defining an exposed or buried residue.
I am assuming it means one of the following:

If an amino acids RSA is below 20% it is buried and above 20% it is exposed. So for an amino acid with an RSA of 21% is considered exposed, this value seems a little low for me. I think statement 2 would make for sense.
If an amino acids RSA is below 20% it is buried and above 80% it is exposed.

Which statement if any is correct?
Paper 1 - see methods section first paragraph
Paper 2 - see figure 5 and table 3
Paper 3 - see abstract and dataset
Paper 4 - see table 2 for total values

Its 1.  Below cutoff, buried, above cutoff accessible.
Paper 1: "A cutoff of 20% was used to define the two states, buried or exposed. With this definition, the dataset was, roughly, evenly split between the two states."
Only two states are possible: solvent accessible and buried.
Paper 2: "A given residue is defined as exposed (e) if its RSA is larger than the cutoff
value, and otherwise it is defined as buried (b)."
Abstract for Paper 3: a cutoff of 20% for two-state definition of solvent accessibility.
If it were def #2 it would be a three state definition.
Paper 4: "On average, 15% of residues in small proteins and 32% in larger ones may be classed as “buried residues”, having less than 5% of their surface accessible to the solvent..."
This paper, by the dean of structural analysis Cyrus Chothia, uses a 5% cutoff, not 20%...
The abstract goes on to say... "The accessibilities of most other residues are evenly distributed in the range 5 to 50%."
This passage hints that SA doesn't even go up to 80%.  Just considering that you often won't get more than say 60% with this calculation.  I'm just guessing; but the thought being that unless you are at a terminus of the protein, which is often disordered and doesn't show up in a crystal structure, you will have two adjoining amino acids for each residue - just the solvent accesible area taken up by the contact with neighbors could easily be 20% of total.

y welcome!  papers are like a wall of words.  This belies the fact that the methods are often the simplest method you can think of.

Are you sure that the RSA formula is right?
I have found a different description：Relative solvent accessibility classes are usually derived from the DSSP program by normalizing it at the maximum value of exposed surface area obtainable for each residue.Different arbitrary threshold values of solvent accessibility are chosen to define binary categories (buried and exposed) or ternary categories (buried, partially exposed, or exposed).
Pollastri, G., Baldi, P., Fariselli, P., & Casadio, R. (2002). Prediction of coordination number and relative solvent accessibility in proteins. Proteins: Structure, Function, and Bioinformatics, 47(2), 142-153.


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What is the difference between average and monoisotopic mass calculations of peptides?
I often have to calculate the mass of peptide sequences using calculators such as here.
I have the option of calculating the average and monoisotopic mass, but what is the difference?

The elements that typically make up peptides and proteins can be present in different isotopes. For example, carbon is present to 98.9% as $\ce{^12C}$, and to 1.1% as $\ce{^13C}$.
The monoisotopic mass assumes that every element is present in the most abundant isotope, the average mass uses the actual distribution of the isotopes in nature.
If you have sufficient resolution in your mass spectrum you can see multiple signals corresponding to different isotope compositions of your molecule.


text:
Why aren't animals diverse in phenotype?
I am not comparing a cat with leopard.
I am just saying that we humans are all one type of creature and we are diverse (I am not saying we are class of mammals and phylum of etc and kingdom etc, because my religion doesn't believe in it).
So consider the class of cats they are one type of species so why aren't they diverse in phenotype like us?
Why do other animals, plants, unicellular organisms not have diversity in their phenotype and how they can recognize each other, like a bird always brings food to his offspring and it can't make mistake by giving it to other offspring of its own species?
So can I say they aren't diverse because of in their meiosis division their chromosomes don't cross over and random assort (alignment)?

Quick note re. face recognition: human face diversity is similar to other species. It seems greater because we have specific sensitivity to tiny details in human faces (as do sheep to sheep faces, etc). Some people lose this through brain damage or from birth (Prosopagnosia), and without it, human faces are as alike as, say, sheep. Try drawing recognisable likenesses of people and you'll see how tiny our facial differences are: caricature artists exaggerate these tiny details massively to make their drawings reliably recognisable.

(sheep make a good analogy because farmers can (with difficulty) learn to recognise some sheep by their faces like how prosopagnostic people can (with difficulty) learn to tell between some faces by focusing on specific details, and there are studies showing that sheep learn to recognise sheep faces with similar speed, ease and duration to humans learning human faces)

In your question, your assumption that animal species are less diverse phenotypically than humans is wrong. I am sure you will appreciate @terdon's answer to this post and @rg255 answer to this post.
Don't forget that we are good at detecting differences among humans (because we evolved for this purpose). We are doing much worse at telling apart animals from other species just because we have not evolved for this purpose. This is the reason why we tend to see human faces when looking at clouds but we rarely see sheep faces! Several studies (here and here) showed that sheep are able to recognize each other (and we even know the number of neurones needed to remember one face). They are probably better at telling two sheep apart than telling two human apart.
Another interesting fact is the so-called cross-race effect. We, humans, are better at recognizing faces of people from our own ethnic group than faces of people from other ethnic groups. For example, a Japanese is very good at Japanese faces recognition but not good at recognizing European faces. Same is true the other way around.
As @user568459 said in the comments: some people are not able to recognize faces. This is due to a cognitive disease called Prosopagnosia (also called face blindness). Those suffering from this disease are not better at recognizing sheep faces than human faces.

So consider the class of cats they are one type of species so why aren't they diverse in phenotype like us?

There is no good definition I think of what is phenotypic diversity (no accurate and objective index to measure it) but at first sight I would tend to think that cats are more diverse than humans. One of the main features one would probably raise when talking about human diversity is skin color. And in terms of color, cats are much more diverse than humans. You may think of an extraordinary diversity when thinking of Norwegian that are taller than Indonesian (I may not have chosen the two extremes) by several centimeters on average but think about cats! The average cat weight 4 to 5 kg but some cats weight less than 2 kg and some other (like the coon cat) weight more than 10 kg (World Record: 21.3 kg). Imagine a human ethnic group that would on average weight 5 times more than another ethnic group! And think also about cats' hair length or tail shape! Humans vary in terms of facial feature (lips size, nose shape, etc.) so do cats. Some look like their face was smashed against a wall while others have a long muffle. Again I welcome you to have a look to this post.

how they can know each like a bird always brings food to his offspring and it can't make mistake by giving it to other offspring of it's own species?

As I said above humans evolved to recognize their own. Many species also evolved in order to recognize their own. In some species individuals use smell rather than visual features in order to recognize each other (odor is also a kind of phenotypic variation). But still some species are poor to recognize each other. For a bird, it seems rather easy to not feed the wrong individual as all their offspring are usually together in the same nest. However you might be interested the lifestyle of the cuckoo who parasites nests of other bird species. Cuckoos' babies and particularly the inner beak resemble to the babies of the species they parasite and often the parents (often the mother only is involved in feeding the young) get fooled and feed the cuckoo.

So can i say they aren't diverse because of in their meiosis division their chromosomes don't cross over and random assort (alignment)?

No, you can't say that! Because they are diverse and because for many of the species you may think about, cross-over does occur. Their genetic diversity as well as their phenotypic diversity is as high than in humans. There is nothing extraordinary about humans (except their brain and the related fact that we predigest our food by cooking it) compare to other lineages. And there is nothing extraordinary to have one extraordinary feature (such as a big brain) that you can't find in other lineages! Many lineages are extraordinary in some sense.

I'd like to add that the variations in individuals within a species are a fundamental observation upon which modern biology is standing.  Darwin wrote at least 2 chapters of Origin of Species demonstrating how animals and plants have a lot of individual variations:

Darwin's argument involved four steps.  First, he noted the wide variation between many types of living organisms: between species of plant, fish, bird and mammal; and also between different family groups within the same species; and also between different exemplars within the same family. Wherever we look - and during his five-year voyage on The Beagle in his early twenties, there were few places on earth where Darwin had not looked - we see differences between organisms: different sizes, different colours, different features, different behaviours and, of course, different survival rates. The theory of natural selection takes variation of species and within species as its starting point.

Before that era (he was not the only one who made this argument) pretty much everyone believed that all animals of a given species were the same.
It took quite a while before everyone was convinced, but an entire generation of scientists was won over.
If you need a modern confirmation of this, look into cattle breeding. Individual phenotypes make some bulls worth many thousands of dollars/euros.  Others nothing. Lastly, now that we understand that the basis of inheritance is in the DNA, we can quantitate the specific mutation rates in various genes and segments of DNA and show that they are comparable from species to species.  These vary somewhat, but humans are not exceptional.


text:
How fast does eyelid close when eyelash is triggered?
What is the reaction of blinking like when an unexpected piece of wood or something hits an eyelash and then an eyeball?
A piece of stone flew into my eyeball when we knocked a wall down, and it hit the white area. Another time, I walked into a piece of wood in the dark, and my eyelid closed faster than I was walking so it protected my eye.
So it got me thinking. Is there a fast neural network in between the lash and the the lid contractors, so that the eyelid can close very fast when the lash is contacted unexpectedly? How fast does the reaction happen?

The human eye is thought to react about 1/24 of a second, but I'm not sure how fast it is when it touches an eyelash

Thought by who? based on what? Please [edit] your posts to conform to the standards of this site.


text:
Removing Sugars Bound to a Protein
I recently purchased some Maltose Binding Protein and as the name suggests this protein binds to maltose. The problem I have is the protein arrive with maltose bound. I know this through native mass spectrometry analysis.
Is there a way of removing the maltose bound to my protein sample while also keeping the protein in its native state?
Examples of two states from the PDB can be seen here: Bound and unbound

It's not clear from the question what the biological source of the maltose binding protein (MBP) is. As you may know E. coli MBP is used as a fusion partner in an expression-purification system marketed by New England Biolabs (this becomes relevant below).
My initial thought upon reading your question was to suggest dialysis. However I found some hints online that this might be problematical and eventually found this on a FAQ at the New England Biolabs site. Looks like this may be your answer.

Question: In order to rebind MBP to the column, the maltose must be removed. Can this be done by dialysis?
Dialysis does not work very well to remove maltose from maltose-binding protein. This is a general phenomenon of binding protein/ligand interactions; after the free ligand is gone, ligand that is released from the binding site usually finds another binding site before it encounters the dialysis membrane (28). We have determined empirically that binding the fusion to a chromatography resin and then washing away the maltose is much more effective. We prefer standard chromatography (e.g. DEAE) as the separation step, since it can separate the Factor Xa and MBP from the protein of interest. In case MBP co-elutes with the protein of interest, we include a large volume washing step to remove the maltose before starting the salt gradient. This way, the mixture can be run over an amylose column afterward if necessary.

So, in summary they are suggesting that you bind the protein to an ion exchange column; wash with lots of buffer; then elute the cleaned-up protein (using salt or a change of pH  - you will have to know something about your protein's behaviour on the column to do this).


text:
Hot water and bacteria
I know that it is common to say, "use hot water when washing your hands" or when you've got a cut, "wash your hands with warm water," etc. I was wondering, why is this the case? Since bacteria grow in warm environments, you would think that it might be beneficial for bacteria. Is this just another myth or is this actually true?

The bacteria wouldn't see any benefit from the warm water in the ~30-60 seconds you're washing your hands, neither would hot water "sterilize" your hands at temperatures you could tolerate. The reason you wash your hands with hot water is because the hot water+detergent soap mix is better for removing oil and dirt than cold water+detergent, which is supposedly where bacteria reside.
Interestingly, there was a study published saying that washing with hot water doesn't have much of an effect on bacterial load. Hot water for handwashing--where is the proof? (Laestadius and Dimberg, 2005). I'd be interested to look more into their methods but the paper is behind a paywall and I don't have access at home.
Also, this paper, Water temperature as a factor in handwashing efficacy by Michaels et al. is open and shows no evidence that hot water is any better than cold water for removal of microbes.

[W]ater temperature exhibits no effect on transient or resident bacterial reduction during normal handwashing with bland soap.

Perhaps warm water just feels nicer while washing, thus encouraging longer washing. I'd suspect this should then increase the removal of microbes.

@edA-qamort-ora-y I'm sure you're right and that this is a published result - I'll have a look if my journal access is working.

The goal of hand washing is to remove surface debris, including foreign pathogens. Most things that most people encounter on a daily basis dissolve more easily in hot water than in cold water. Thus, hot water around 100° F is used to facilitate this debris removal process, although the ideal culture temperature for E coli and a raft of related bacteria is around there too.
That being said, from the wikipedia on hand washing:

Contrary to popular belief however, scientific studies have shown that using warm water has no effect on reducing the microbial load on hands

"Everything that can dissolve in water dissolves more easily in hot water than cold water." That's not actually true. Gasses are much more soluble in cold water but even some solids, such as sodium sulphate dissolve better in cold water.  (I concede that the removal of adsorbed gasses and sodium sulphate are not major factors in hand-washing. :-) )

@DavidRicherby I changed the wording a little to reflect your comments, but I am curious about what you said. Do you have a reference?

@DavidRicherby I would also like to see any source for a solid dissolving better in cold water

http://2012books.lardbucket.org/books/principles-of-general-chemistry-v1.0m/s17-04-effects-of-temperature-and-pre.html
The curves with a negative slope are the examples you seek.

@user3790338 Just Google "sodium sulphate solubility". One example is the curve at Wikipedia which shows decreasing solubility from 38C to about 120C (presumably under enough pressure to keep the water liquid at those temperatures).

Another indirect but common example of a solid being more soluble at low temperatures is calcium carbonate, which is only weakly soluble in pure water but with carbon dioxide dissolved in the water the solubility increases.  Remove the CO2 by heating (remember gas solubility decreases with increasing temperature) and the CaCO3 precipitates out.

It makes no difference at all. To sterilize your hands you would need to wash them with water at around 115 degrees C at 1.2 atmospheres of pressure for over 30 minutes – that's obviously not going to happen. The bacteria that normally reside on your skin can easily tolerate tap water, no matter whether the faucet is set to maximum hot or maximum cold, or just average. Even if the temperature of the water could appreciably slow down bacterial growth (which it can't), once you've washed your hands your skin temperature goes back to as it was before the washing in a minute or so.

Besides that: The bacteria living on our skin are usually not threat for us and belong to our normal bacterial environment. We learned to live with it and to control it.

The fact that you can't wash your hands in water hot enough to sterilize them doesn't mean that hot water has no benefits. (Though the other answers suggest that it doesn't.)


text:
Blood cells penetration
Is there a type of blood cell that can reach all other body cells? By "reach", I mean to "touch" the surface of the target cell.
If we look at the red blood cell for example, that moves in blood vessels, there are cells close by - after the wall of blood vessel - that get oxygen by diffusion and not by direct contact, as far as I know.

Drugs are usually not recognized by the immune system. Can you explain further, why you want to do this? And which disease you want to target?

Usually but there are some that may lead to immune response: http://en.wikipedia.org/wiki/Idiosyncratic_drug_reaction Also it is a way to improve targeting, theoretically. I added a link in the question about using of Red blood cells. My question more about the type of cells that can reach all the cells and organs in a body. Thank you.

That's why I was asking what disease you want to target. You not only have to get the drug inside you transporter cell, you have also get it out again on the right place (and presumably only there).

You right, Chris, it's very theoretical. There are no some special disease. I was thinking about using cell like "inside doctor". The cell will recognize the target and make some action. So need to chose some cell to start from it. This cell need to reach  all the cells in the body and about that is my question. How to teach the cell to do it, is much much complicated =) Just wondering if there is cell that reach all the places in the body (or most of them).

Why not use a PEGylated liposome or something to coat the drugs in a protective layer? You could add a targeting ligand to the surface to help direct it toward receptors at the target site and not need to rely on getting into and out of transport cells. Also, there is quite a bit of existing research to build on.

thanks you, but I just want to know  if there is some type of cells that can reach all the places in the body (or most of them).

Basically all circulating blood cells have approximately the same chance of reaching a target tissue. That's what blood is for, to release oxygen and nutrients to every cell in the body, and to pick up waste products. If plain old blood were unable to do this, the tissue would die. That's called ischemia. Sure, cytotoxic T cells have access to all the cells, but don't act independently of other immune processes. But you can choose other cells as well.

I don't thinks that it is true. For example Cancer cells don't get enough oxygen because red blood cells don't reach them, there is no blood vessels near them. So all the oxygen that they get is from diffusion through neighboring cells.

That's outright nonsense, and it doesn't belong here or anywhere else. If what you said was true, cancer cells would die and we wouldn't have to worry about cancer at all. Instead of guessing, why don't you do some actual reading? You can start with your own error. Here's a clue: Molecular mechanisms of tumor vascularization.

The amount of mitochondria in cancer cells is often very little. It's true that they create a blood vessel in the end. But there are stages that the main source of energy is  pyruvate - lactate. But again. That not what I am asking. As far as i know - red blood cells not touch all the cells in the body. They move mainly in blood vessel and not between the cells.

No, there is not. Cells in the bloodstream reach most areas in the body, but there are certain privileged areas which stop cells, drugs and macromolecules from leaving the bloodstream and entering the tissue. These privileged areas are thought to exist in sensitive or important areas for their own protection:

Immune privilege is thought to be an evolutionary adaptation to protect vital structures from the potentially damaging effects of an inflammatory immune response. (Wikipedia)

The most common example of such an area is the blood-brain barrier, but there other barriers to privileged areas, including the blood–testis barrier, the blood-retinal barrier and the blood-ocular barrier. Cells do not pass from the blood through these barriers, except in disease (Larochelle et al. 2011).

Related reading:
The first chapter of Blood Groups and Red Cell Antigens has a decent high-level overview of different types of blood cells and their roles in the bloodstream and the immune system.
How do immune cells overcome the blood–brain barrier in multiple sclerosis? Larochelle et al. 2011

Thank you! But in wiki - "Antigens from immune privileged regions have been found to interact with T cells in an unusual way" - that mean that T cells reach the region, just don't work.

That actually means that antigens that escape from the privileged regions (usually due to injury or illness) are recognized by the immune system in ways we don't understand. This interaction is not a normal occurrence. Reading further, you can see that there are immune cells in some of those areas (i.e. eye), they just don't move back and forth


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How does the genetic material differ between dizygotic twins and ordinary siblings?
I am going to give a rough presentation on twin studies.
The terms heritability and concordance are included already, but I struggle with understanding the difference of the genetic material of dizygotic twins and ordinary siblings.
While ordinary siblings evolved from different sperm and egg cells, I only know that dizygotic twins form when two different sperm cells fertilize egg cells of the same cycle. However, I'm not sure if these cells are identical products of the same meiosis process.
I appreciate your help and time.

Differences in genetic material between dizygotic twins and siblings are likely to be very similar.

Dizygotic twins, or so-called fraternal twins, on the other hand, are no more genetically similar than any other set of siblings. - Utah.edu

Since all oocytes are all synthesized during fetal development, dizygotic twins are no more likely to come from a single mitotic division than siblings are.
Also note that in human females, only one egg comes from the a full mitotic division:


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Anopheles arabiensis lifespan and sporogony duration
What is the longest observed lifespan of a mosquito, especially of the species Anopheles arabiensis? How long does it take for Plasmodium parasites to develop and move into the salivary glands of these mosquitoes?
I need this information to calibrate my model on malaria transmission.

What is the longest observed lifespan of a mosquito, especially of the species Anopheles arabiensis?
Mean lifespan calculations for Anopheles arabiensis range from 14 days (Karoki, 2013)* to 21 days (Yamada et al. 2014)**
*This Masters thesis tests efficacy of different drugs against An. arabiensis, 14 days is the mean control lifespan.
**These An. arabiensis are of a specific strain but no indication is given that the life span is different than normal An. arabiensis.
The CDC Anopheles mosquito page is another good source for information on Anopheles life spans:

The adult females can live up to a month (or more in captivity) but most probably do not live more than 1-2 weeks in nature.

How long does it take for Plasmodium parasites to develop and move into the salivary glands of these mosquitoes?
US National Institutes of Health:

Growth and division of each oocyst produces thousands of active haploid forms called sporozoites. After 8-15 days, the oocyst bursts, releasing sporozoites into the body cavity of the mosquito, from which they travel to and invade the mosquito salivary glands.

United States Air Force (USAF):

The length of the developmental stage in the mosquito not only depends on the Plasmodium species but also the mosquito host and the ambient temperature.  This may range from eight days in Plasmodium vivax to as long as 30 days in Plasmodium malariae.

Wikipedia (Plasmodium falciparum) :

Over the period of a 1–3 weeks, the oocyst grows to a size of tens to hundreds of micrometres... The sporozoites then migrate to the salivary glands and complete their differentiation. Once mature, the sporozoites can proceed to infect a human host during a subsequent mosquito bite.

US Center for Disease Control:

After 10-18 days, the parasites are found (as "sporozoites") in the mosquito's salivary glands. When the Anopheles mosquito takes a blood meal on another human, the sporozoites are injected with the mosquito's saliva and start another human infection when they parasitize the liver cells.

Overall, it depends who you ask, but especially on the species of Plasmodium you're working with. I'd look for specific papers on the development cycle you're modeling and run with it.


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Are there any effects of elevated Cysteine levels on cognitive function?
I'm looking at this diagram of homocysteine metabolism and see two distinct pathways that the amino acid may get metabolized to: with vitamin B12 it gets converted back into methionine, while with B6 it gets converted to cysteine.
I've followed the links for the products that Cysteine gets metabolized to and could not find any references to cognitive effects. Are there any cognitive effects of Cysteine or other amino acids that it gets metabolized to?

I'm interested in learning what are the possible effects of pharmacological (50mg - 100mg) doses of vitamin B6 on this metabolic cycle, while folate and B12 levels stay at the baseline levels. It seems to me that if vitamin B6 is more readily available, there will be a higher chance of homocysteine metabolized into Cysteine. Is this assumption correct? What are the implications to this metabolic cycle that may be caused  by elevated levels of vitamin B6, while keeping folate and B12 at baseline levels?
I'm particularly interested in the implications of this process on dreaming. There has been numerous reports among dreamers that 50-100mg of vitamin B6, taken in the 3rd-5th sleep cycle produces very intense and vivid dreams, sometimes featuring spontaneous awareness (lucid dreams), while B-complex vitamins (B6, B12, Folate, Niacin, etc) appear to produce the opposite effect - very vague dream recall.
I'm trying to understand if there is indeed a connection between the metabolic pathway that homocysteine may take and the vividness of dreams, capacity to recall them clearly and the phenomenon of being able to spontaneously realize the fact that the person is dreaming while still asleep.
Thank you for your input!
Diagram obtained from here

Thank you for that great article. I've read it and see that they are using all 3 vitamins, although the B6 levels are indeed elevated 1000% (20mg/day for 2 years) but the B12 levels are 5000% daily recommended dose, and folic acid is 2000% daily recommended dose.

Your shown homocysteine pathway is way too simple. Especially the production of the neuromodulator H2S from excess sulfur amino acids is not shown. In the last years many more enzymes and reactions have been discovered. I have summarized human sulfur amino acid metabolism in reactome.org, so please use this link to discover all the details and new papers. I'll also append some of the relevant papers below.
Brosnan, JT, Brosnan, ME The sulfur-containing amino acids: an overview 2006 J Nutr PMID 16702333
Remethylation of homocysteine to methionine can also happen using betaine as a methyl donor. This reaction is also part of choline catabolism.
Li, F, Feng, Q, Lee, C, Wang, S, Pelleymounter, LL, Moon, I, Eckloff, BW, Wieben, ED, Schaid, DJ, Yee, V, Weinshilboum, RM Human betaine-homocysteine methyltransferase (BHMT) and BHMT2: common gene sequence variation and functional characterization 2008 Mol Genet Metab PMID 18457970
Bearden, SE, Beard RS, Jr, Pfau, JC Extracellular transsulfuration generates hydrogen sulfide from homocysteine and protects endothelium from redox stress 2010 Am J Physiol Heart Circ Physiol PMID 20817827
Dominy, JE, Stipanuk, MH New roles for cysteine and transsulfuration enzymes: production of H2S, a neuromodulator and smooth muscle relaxant 2004 Nutr Rev PMID 15497768
Stipanuk, MH, Ueki, I Dealing with methionine/homocysteine sulfur: cysteine metabolism to taurine and inorganic sulfur 2011 J Inherit Metab Dis PMID 20162368
Chiku, T, Padovani, D, Zhu, W, Singh, S, Vitvitsky, V, Banerjee, R H2S biogenesis by human cystathionine gamma-lyase leads to the novel sulfur metabolites lanthionine and homolanthionine and is responsive to the grade of hyperhomocysteinemia 2009 J Biol Chem PMID 19261609


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What causes random long white body hairs?
I'm sure many of you have experienced this - you scratch your back or brush your hand over your arm and find a ridiculously long thin white hair, sometimes as long as 3 or 4 inches. I know a few people who get these quite frequently, and anecdotal evidence seems to suggest they almost grow almost overnight. A quick Google search for "single long white hair" throws up plenty of references to them, and various beauty advice, but I'd like to know the biology behind them.

What triggers abnormal body hair growth?
What causes these hairs to grow so much longer than other "normal" body hairs in physical proximity?
Why are they so thin and white compared to other body hair?

My primary hypothesis is that the hair cell grows rapidly in an uncontrolled manner, similar to how cancer might, and that the unusual appearance is due to the cells being starved of nutrients. I may be completely wrong, though.
I'm mainly interested in humans here, but if more in-depth research is available on animals that'd be interesting too.

I can add that this has happened to me.  I actually hadn't ever heard of it happening to someone, and I thought maybe somehow someone else's hair became embedded in my skin!  I seem to recall it being very long as well--three or four inches in my arm that just showed up one day.

Would this need to be because of "damaged" DNA or could it just be a repressed gene like the "one white tulip in a field of red" phenomenon? I apologize ahead for terminology error.

I beg your pardon for being skeptic, but apparent overnight growth may also be explained by just being unnoticed for weeks or moths. They only seem evident after being noticed, but before that long hairs are not so conspicuous. I can remember finding a long hair, forgetting about it and finding it some days later.

Minoxidil use might complicate a naturalistic answer to the question.

Since it's been so long, I guess a rushed speculative answer might be at least an idea.
DNA gets damaged randomly all the time, and repair mechanisms are in place to fix it. When the damage is too large or of a very complex kind, permanent mutations can develop, and cause disorders such as cells proliferating without control - i.e. tumours.
I could imagine that the same mechanism could result in cells which overproduce keratin (hair) without melanin (pigment), if the associated genes are affected. Considering that trichocytes and melanocytes, the two cell populations at the base of the hair responsible for production, are among the most rapidly proliferating cell types in humans, an increased risk for mutation would not surprise me. I was able to find a few papers on keratin-associated gene clusters by a quick search, but they all seemed to be related to production of fragile hair rather than excessive length.

Melanoma is not the most common type of skin cancer.  Squamous cell carcinoma and basal cell carcinoma are much more common, though with a much better prognosis.

Silly me, thanks for the hint, goes to show the frequency with which a condition is mentioned (or seems to be) doesn't always correlate with its importance.


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Predictable microchimerism
I read in New Scientist recently that microchimerism occurs between previously born siblings and grandparents, not just the mother.
Do we know which parts of the genome are likely to be transferred? Is it possible, given a genetic sample of relatives, to predict which genetic traits might be transferred by microchimerism?

Microchimerism, as defined by wiki, is the presence of a small number of cells in an organism that are genetically distinct, and originated from a separate organism. The example they give is an unborn child and the mother can 'swap' immune cells, and retain these for many years.
Your question about which parts of the genome are transferred is therefore not really valid, because the cells are transferred, rather than bits of genetic information. Therefore 'traits' are not transferred either, but distinct immune cell lineages can be transferred and persist - this can confer an immune advantage, so this 'trait' could be said to have transferred, but no genetic information has been swapped by the cells.
Although it does not say it on wiki, this presumably only applies to cells of the same species, but a different organism, otherwise infections would also count.

Update 20th July 2012
The New Scientist article you have linked to in the comments can be found online here.
It says that we may be more microchimeric than we imagined, because a recent study in Blood found that half of the mothers studied were positive for male T-cells (Dierselhuis, 2012). The authors note that it is remarkable that the immune cells from neither the parent or the offspring attack the other, and that this may explain the observation that siblings make better donors.
Another finding that mothers are found positive for cells from their own mothers, and even their own grandmothers, and that the cell numbers increase during pregnancy (due to clonal expansion of the inherited immune cells) reinforces the opinion that microchimerism is commonplace (Gammill, 2011).
So to reiterate, immune cells can be inherited from your mother, and she may also end up with leukocytes from her offspring, which can in turn be passed on. This is a very fascinating way in which the immune system seems even more complex and helpful.

This seems to be counter to what the article I read said. They indicated that genetic material from siblings and a grandparent was found in children.

In a sample of the blood it is not uncommon to find fragments of DNA. These may well be from a parent or grandparent, but the DNA (as I understand it) is 'left over' from (e.g.) an apoptosis event, rather than found in the host cells. It is also possible to find fragments of bacterial DNA in circulating blood, but this does not imply that the DNA has been incorporated into the hosts, and therefore no traits have been transferred.

I remember it mentioning increased risk of transfer of diseases, so I'll dig out the article tonight and post an excerpt.

@Polynomial I'd be interested, too.  I did a fairly extensive search and only found a NS article on Marmosets from 2007.  Is that the one that you found?

@jonsca Nope, this was recent, perhaps a month or so old. I completely forgot about this last night, but I'll try to remember to dig out the article tonight.

@Polynomial my apologies if I have misinterpreted your question! A link to the article would definitely help resolve this :) thanks.

Found the article! It's in edition #2869, on page 12. Got a jpeg of it here.


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When are a female's eggs created?
Since a human woman is born with a set number of eggs, and meiosis is the process by which sex cells are produced, would meiosis in a woman happen before she is born or soon after she was born?
If meiosis creates sex cells and women are born with a set number of sex cells, when in the development of a woman are her sex cells created?

I can't understand your question.

@WYSIWYG If meiosis creates sex cells and women are born with a set number of sex cells, when in the development of a woman, are her sex cells created?

Oocytes, or immature female eggs, develop in the fetus's ovaries during pregnancy. This graph (U. New South Wales) shows the oocyte population over time in a human female:

Although the x-scale is kind of confusing (months when negative, years when positive), you can see that the fetus has all the oocytes it will ever have at the peak 18-22 weeks after conception. Since age 0 is birth, meiosis in a woman happens long before she is born.

Meiosis starts before birth but finishes after.


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Flicker fusion threshold for insect eyes
I searched on bio-numbers for flicker-fusion threshold of insect eyes in general and for flies in particular. Ruck, 1961 gives values for some flies. Is there any database maintained for these values? Is there any comparative study done for many flies/insects?

"The flicker fusion frequencies of six laboratory insects, and the response of the compound eye to mains fluorescent ‘ripple’" (Miall, 1978) touches on flicker fusion thresholds for six different insect species commonly used in laboratory settings:

Locusta migratoria, Periplaneta americana, Saturnia pavonia, Antheraea pernyi, Glossina morsitans and Drosophila hydei.

I also found this paper: Potential Biological and Ecological Effects of Flickering Artificial Light (Boyles, 2014) that includes an exhaustive literature search of flicker-fusion thresholds and other related data for many species (including insects). If you're looking for an authoritative source, I'd say that this is it (as there doesn't seem to be a database with this information).


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How can 3' end polyadenylation protect cellular RNA from ribonuclease degridation?
In this link, it states:

It is worth noting that all intracellular RNAs are protected from RNase activity by a number of strategies including 5' end capping, 3' end polyadenylation, and folding within an RNA protein complex (ribonucleoprotein particle or RNP).

I do not understand how polyadenylation protects the intracellular RNA from ribonuclease. I always thought that polyadenylation was a way to mark RNA for degradation, not protect it from degradation. Perhaps it is speaking of protection from exoribonuclease only and not endoribonuclease?
Can someone explain this?

Actually, whether polyadenylation protects an mRNA or makes it susceptible to degradation depends on the organism. From Dreyfus and Régnier 2002:

In eukaryotes, poly(A) tails usually act as stabilizers of intact mRNAs, whereas in E. coli they serve to accelerate the destruction of fragments.

This is due to different protection mechanisms in prokaryotes and eukaryotes:

The Poly(A) Binding Protein (PABP) inhibits deadenylation in mammalian cell-free assays. Although poly(A) binding proteins exist in E. coli as well and although they can protect poly(A) tails against PNPase in vitro (Feng et al., 2001), they may be unable to exert a significant protection in vivo because poly(A) tails never grow long enough to enable them to bind.

Important to note is that bacterial mRNA degradation usually starts internally, in contrast to eukaryotic mRNA degradation which happens (exonucleotically?):

As noted above, another distinctive feature of bacteria is the fact that polyadenylation usually affects the degradation of fragments only, because the initial attack on most intact mRNAs is endonucleolytic.

All of these quotes come from the review I referenced at the top of my answer, I recommend reading it as it's very comprehensive (and interesting).

According to my book Molecular Biology Principles of Genome Function by Craig et al, eukaryotic RNA degradation does not have an initiation, as in bacteria where pyrophoasphate hydrolase hydrolyses the 5'-triphosphate to 5'-monophosphate, and is therefore the initiatior.
In eukaryotes the polyA-tail is shortened by a deadenylase enzyme complex which contains a 3'to 5' exonuclease called CCR4. But deadenylation of polyA-tail doesn't necessesarily need to lead to degradation since it is reversible by post-translational readenylation.
Poly A tail protects the mRNA by not letting the nucleases reach the mRNA itself. As long as poly A tail is degraded, its just fine, since it can readenylate back again. The figure below shows the degradation of mRNA in eukaryotes, which can be either in 5' to 3' direction or 3' to 5' direction.

What you are thinking of is ubiquitination of proteins, which is a post-translational process which can mark the proteins for degradation.