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How can I download a gene sequence from GenBank (NCBI)?
Could you tell me the steps to find and download a gene sequence from GeneBank?
I would appreciate your help.

I think you can very easily find the steps for this with a little research.

Go to the NCBI website, fill the search field with relevant information in the upper part of page, select "nucleotide" from the drop-down menu just to the left of the search field and click search. You will get a list of items, by clicking "FASTA" link below any of them will bring you the corresponding sequence.
Depending on your interest, you can choose other databases from that menu, and some of them are interlinked. For example you can do the search by selecting "gene" instead of "nucleotide" and when displaying a selected gene info, you can see the links that lead to nucleotide database for getting the sequence.


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Why would eating the meat of cows fed with antibiotics trigger an immune response to the antibiotics?
The document "Antibiotics and Antibiotic resistance" contains the following paragraph:

The indiscriminate use of antibiotics in feed stuffs means that humans
  may receive unwanted doses of antibiotics in meats, eggs and milk.
  This exposure may cause the development of an immune or inflammatory
  response to the antibiotic so that the human cannot be treated with
  the drug at a later date. The practice has also resulted in the
  development of antibiotic-resistant strains of bacteria.

Here are my two questions that test general biology principles:

How can there be an immune response to an antibiotic?
I think that the answer to the first question will answer this one anyways, but for the sake of completeness: why would the immune response to an antibiotic prevent the antibiotic from being used again? (I may know how to answer this once I actually know what an immune response to an antibiotic looks like.)

How can there be an immune response to an antibiotic? Because it is a foreign molecule. So people can develop an allergy to it (there are quite a lot of people allergic to penicillin and it's derivates).

So do antibiotics count as antigens in that sense? I believe that antigens are any substances which have the capability of triggering an immune response...

The antibiotic or some product of metabolizing it might be an antigen.

It's difficult to know how to answer this question because it is unclear why you think that this might not happen.
As skymningen has said in comments, any foreign molecule is capable of acting as an antigen,  in principle. We know that people develop allergic reactions to penicillin. And if you do a Google search for immunoassay [insert name of antibiotic] you will probably find results indicating that there are commercially-available kits for assaying the antibiotic based upon antibodies directed against it.
It is however worth noting that these anti-antibiotic antibodies were certainly not raised directly by feeding, or even injecting, the antibiotic. This is because the immune system doesn't really respond to small molecules. If you want to make antibodies directed against a small molecule you have to tag it on to a protein - this is called haptenisation. Then some of the antibodies that develop against the modified protein will recognise the hapten (the small molecule). Another example of this is the development of immunoassay for steroid hormones.
So we have to imagine that a fed antibiotic enters the bloodstream and then reacts chemically with a blood protein - essentially the antibiotic has haptenised itself. I seem to recall reading that β-lactam antibiotics are fairly reactive against lysine residues. The propensity of different antibiotics to provoke an immune response may be simply a function of their reactivity.

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No digested product bands, but markers are visible. What could be the reasons?
I had to digest my insert containing plasmid and another vector into which my insert has to be ligated, each with BamHI and NotI.
Upon digestion, I ran the gel to check for results. The digestion seemed to be ok the first time with reasonably good bands. Since I needed more concentration I ran the gel with the same reaction setup (30microL) for the second time.
Well 1 - 100bp marker
Well 2 - Insert in a vector (820 ng/microL, 3microL)
Well 3- Plasmid vector into which the insert has to be ligated (1000 ng/microL, 2microL)
The marker bands were faint while there were absolutely no bands in the other wells. What could be the reason for failure during the second run? I've performed the second time digestion in a span of 40 min after the first one.

Usually there is no issue of mixing. One possible reason is degradation. But you say marker is faint (marker wont get degraded). Stain the gel with extra EtBr and see again.
Was it the same gel?? (If so then definitely stain it again beccause EtBr migrates towards the cathode)

Can you show the gel image? Otherwise I support WYSIWIGs idea. There might not be enough EtBr left in the gel. What bands of the marker are visible? All, or only the highest? Additionally: Are there marker bands visible in the same size region as you digested DNA? If so, your problem is on the digest side. You are loading a ton of DNA on the gel, this should be visible.

Ethidium will also break down slowly under fluorescent lights. It's usually not a problem, but I did an internship at a startup so poor that they reused the same gel over and over, so we kept it covered in aluminum foil and had to restain it all the time. But it looks like you're using enough DNA to see. Make sure the EtBr and gel and buffers are fresh.

@user137..EtBr doesn't get degraded soon.. It diffuses/moves out of the gel.

Yup, it looks like the DNA degraded. Because my first gel run gave expected results. What is surprising is, this never happened to me. I used the same reagents and protocol. I am unable to understand what caused the degradation. If any of the samples or reagents is contaminated with DNase, I'll have to dispose all the stock and prepare fresh one now. But, how do I know what caused this? Could one of the causes be voltage too?

@WYSIWYG yes, it was the same gel. I've disposed the gel already. I want to try it again, I've got enough samples left with me. But, if the problem is degradation, I should be able to identify what is causing this.
I've gel image, with nothing but marker bands. All the bands of the marker well are visible.

Usually there is no issue of mixing. One possible reason is degradation. But you say marker is faint (marker wont get degraded). Stain the gel with extra EtBr and see again. If you reused the gel then definitely stain it again because EtBr migrates towards the cathode.
It doesn't seem like degradation to me but to be cautious for the next time:

Use freshly autoclaved water. If you are not sure then filter using syringe and keep some 10ml for yourself
Ensure that the plasticware are also autoclaved properly.
Use endo - plasmid extraction kits, if available
Dissolve the plasmid in TE (only if conc is high and you need not put much in the reaction mix else EDTA interferes in the reactions. In that case use Tris-Cl pH-7.6 ).
If you have to digest a lot of DNA then set up small reactions in many tubes.
Do not incubate the reaction for a very long time (>6h).
Inactivate the enzyme by heating if you are not running the gel immediately.
Ensure that the gel running buffers are not too old.

Agreed, seems like overall signal is week, so a stain/destain seems in order

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Explanation of the terms "downstream signaling" and "upstream signaling"
In molecular biology, what's the meaning of the terms "downstream signaling" and "upstream signaling"? What's the difference between them?

You are thinking of transcription factors? Or is it more signal transduction?

@Chris well, I'm not sure. I presume that one uses the same terms in both contexts because they have something in common, right? I'm looking for an explanation of these terms in their more common uses.

It simply means after and before with respect to the flow of information in a given pathway. For example, consider this schematic representation of a pathway:
TF1 ==activates==> gene1 ==produces==> Kinase1 ==phosphorylates==> ProtA

In this schema, Kinase1 is downstream of TF1 and upstream of ProtA. Or, to take a classic example (source):

MEK1/2 is downstream of Raf and upstream of ERK1/2.

Perhaps worth pointing out that there is a flow of information in a signalling pathway so at any point in this flow you can look upstream, to where the information came from, or downstream to where it is headed. That's where the metaphor comes from.

If you think of a transcription factor, than this factor needs to be regulated. This happens either by signalling pathways and also on the gene expression level (how much of the factor is available). So everything what regulates this factor is called upstream (look at the image below).
Everything which is regulated by our factor (other genes) is located downstream of it. I think these terms come from the flow diagrams which are pretty usual in this context.
Look at the figure (from SABiosciences)

Here the signal comes from the membrane and is mediated through different factors until its passed into the nucleus.


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Why cytotoxic T cells don't kill dendritic cells when they present antigen?
When a cytotoxic T cell (CTL) recognizes a peptide presented in the MHC-1 of a dendritic cell (APC), why it doesn't kill this cell?
I know that initially, in the lymph node, the T cell is inactivated. But eventually it becomes activated and travels to the tissue, where it again finds APCs presenting the offending antigen. So why doesn't the CTL kill the APC at this point?
Moreover, not all MHC-1's of the dendritic cell present antigens from phagocytized cells. Some MHC-1's present peptides coming from the dendritic cell itself, so that if the dendritic cell itself becomes infected with virus or bacteria, it can be killed by a CTL that recognizes the peptide. So in this case, the CTL does indeed kill the dendritic cell.
So my question is, when exactly does the CTL kill the APC? How does the CTL knows that should or shouldn't kill the APC?

Afaik. if the CTL is not activated yet, it requires a verification signal from a HTL or a very strong stimuli from the APC. Now I don't know yet what happens when it is already activated and meets with an APC presenting the same peptide. Afaik APCs do present the antigens on MHC2 and MHC1 as well, and it does not depends on whether the cell is infected, so I guess there is a way to prevent cell death in these cases. It probably depends on the amount of TCRs activated and other signals as well e.g. stress signals of the APC or cytokins.

Effector T cells have lower TCR stimuli threshold for proliferation than naive T cells. I haven't found any article about killing TCR stimuli threshold and about what environment is required for successful effector function by cytotoxic T cells. I found that by HIV not infected CD4+ T cells can be killed accidentally by effector T cells so I think the APC can be accidentally killed by the T cells too and the outcome depends on the strength of the TCR stimuli, and other environmental factors (costimulation). It is not clear for me yet how naive and effector T cells differ by activation.

The presentation of antigens on the cell surface is done by two different molecule complexes: The MHCI and the MHCII complexes. These are coupled to different pathways and are recognized by different T cell populations.
The MHCI molecule presents all proteins which are present in the cell on the surface. To do so, peptides coming from the proteasome (where long proteins are digested) are transported in the the endoplasmatic reticulum (ER) by a special transporter called Transporter associated with Antigen Processing (TAP). By doing so, a profile of all proteins expressed in the cell is presented on its surface, including proteins which arise due to an infection of the cell. MHCI is recognized by cytotoxic (or CD8 positive) T cells which subsequently kill all cells which show foreign antigens on their surface (since this is a sign for an infection).
The MHCII complex only presents proteins on its surface which have been taken up by phagocytosis (so here only external proteins are presented). To prevent binding of cellular proteins in the ER (like for MHCI), the binding pocket of the receptor is blocked by the invariant chain. This chain dissociates only when the MHCII complex enter the phagolysosome with its acidic conditions. This makes sure that only proteins which have been taken up by phagocytosis are presented. MHCII complexes are recognized by helper (or CD4 positive) T cells, which in turn can activate B cells to raise a proper immune response.
In theory this should prevent all problems. In the practical life this is different for dendritic cells in a mechanism called "cross presentation". In this mechanism antigens which are taken up by phagocytosis are exported from the phagolysosme and imported into the ER. This pathway also involves the TAP protein. By choosing this way, APC can activate naive CTL which will kill all cells which present this antigen. See references 1 and 2 for more details.
This pathway is important for the immune response to a number of viruses as well as tumors which evade the MHCII mediated immune response by suppressing the MHCII antigen presentation. See references 3 and 4.
It is also important for the stability of the self-tolerance by the deletion of auto-reactive CTL. See reference 5 for details.
References:

Outside looking in: the inner workings of the crosspresentation
pathway within dendritic cells
Cross-talk between the endocytic pathway and the endoplasmic
reticulum in cross-presentation by MHC class I molecules
Role of bone marrow-derived cells in presenting MHC class I-restricted tumor antigens.
Cytotoxic T-cell immunity to virus-infected non-haematopoietic cells
requires presentation of exogenous antigen.
Exogenous class I-restricted cross-presentation of self antigens can
lead to deletion of autoreactive CD8+ T cells.

The question was about what happens if an effector CTL meets with an APC, which presents a peptid from the proper pathogen. Why does the CTL not kill the APC? Note that APC can present pathogen peptides both on MHC1 and MHC2 without becoming infected by the pathogen. So I don't think this answers the question...

I agree with @inf3rno. I don't see how this answers my question.

I misunderstood the question and have adapted it now. Thanks for the notification.

The adaptation is in the last paragraph?

You might find this paper helpful in answering your question. It proposes that Memory CD8+ T-cells protect dendritic cells, with TNF-alpha playing a major role in the molecular mechanism of doing so
Watchmaker, P. B., Urban, J. A., Berk, E., Nakamura, Y., Mailliard, R. B., Watkins, S. C., … Kalinski, P. (2008). Memory CD8+ T Cells Protect Dendritic Cells from CTL Killing. Journal of Immunology (Baltimore, Md. : 1950), 180(6), 3857–3865.
It can be found at:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2905219/

Here's couple links explaining this:

Allison himself talks about the role of CTLA-4:
https://www.researchgate.net/publication/327062074_Fundamental_Mechanisms_of_Immune_Checkpoint_Blockade_Therapy
Clear explanation of what happens (see Figures 1&2):
https://www.researchgate.net/publication/329214140_CTLA-4_and_PD-1_Control_of_T-Cell_Motility_and_Migration_Implications_for_Tumor_Immunotherapy

Briefly, CTLA-4 is expressed shortly after T cell activation and serves to protect APCs (and everything else) from excessive T cell activation and proliferation. Also CTLA-4 makes T cell detach from APC and travel to where it's action is needed - into inflammatory sites.

Welcome and thanks for your answer. We generally encourage more elaborate answers, but because of the referencing a +1 nonetheless :)

Here is some exciting textbook reference (Janeway 5th ed.):
https://www.ncbi.nlm.nih.gov/books/NBK27118/
See Figure 8.14 and compare with text. Whereas the figure shows some presentation of antigen at the site of infection, the text does not explicitly refer to the figure of the site of infection, and says that presentation of antigen happens only after (maybe during) movement of that same APCs from the site of infection to the lymph nodes. It seems to be only there that presentation takes place (and, presentation by dendritic cells means priming, defined by environment of lymph node,  in contrast to presentation by infected non immune cells).
My point is that presentation does NOT happen at peripherical sites of infection which is contrary to the statement in  your question. According to Janeway's wording - taken literally  - before priming in lymph nodes has taken place there is no danger to dendritic cells in presenting at the site of infection. Successful presentation in lymph nodes thus becomes a prerequisite for any killing of  presenting APCs. This looks like some self-regulatory mechanism to me.
It's a different question if, while protecting during the priming process,  the "known" (your question is based on the premise that there is no killing during priming) second signal B7   in presentation induces tolerance for a certain time period. Again, according to basic textbook wisdom, at lymph nodes "presentation is priming". Presentation at site of infection by APC is no priming. Priming means that t-cells are given permission to divide. It is not trivial to see: APCs, dendritic cells, must be protected in lymph nodes "only", as it is "at that point" they are doing their job of priming. When they present the antigens in the lymph nodes they meed naive T-cells that have yet to become cytotoxic - which implies a time lag for cell division and expansion of the t-cell clone. Before naive t-cells have divided and differentiated there can be no killing. The effet of B7-receptor might last that long - within that time lag a few other specific t-cells might presumably have been primed, then the APC might simply not be needed any more.
Some references
Question of  S. Oncosuresh at researchgate at  https://www.researchgate.net/post/Why-Dendritic-cell-are-not-killed-by-T-cells-when-they-present-antigen
It is the "speculative" answer by Ye Tian from the University of Chicago which I feel inclined most to: cp. "... if by then" (they will kill the DC's presenting).
You say, quote: "... and travels to the tissue, where it again finds APCs presenting the offending antigen."
It's a possibility that the assumption that there is presentation by dendritic APCs at site of infection is wrong.
In that case the following reasoning applies:  If there is presentation at site of infection presenting dendritic cells in fact are being "simply" killed as they are not needed any more, as the very fact that they meet primed(!) cytotoxic t-cells shows that presentation in lymph nodes for purpose of priming has succesfully been undertaken "already".

Major reason – Helper  T-cells induce APCs to produce an endogenous granzyme inhibitor (serpin SPI) by APCs. So APC's are protected from being killed (which normally involves granzyme-induced apoptosis due to perforin release by the cytotoxic T-cell)
Also, downregulation  of  cell  surface  expression  of  the CCR7 homing receptor on activated cytotoxic T-cell.  Not attracted to APC.

I presume these things (overexpression of the SPI and downregulation of the CCR7) can also be performed by clever malignant cells. Are there examples of this?

What''s your reference on this?

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Transcriptionally-mediated DNA damage
I'm researching the genetics of brain cancer, and finding a huge number of mutations in voltage-gated channels.  It stands to reason that some of this DNA damage is due to the DNA being transcribed heavily, or in an open chromatin conformation more often, leading to more breakage and damage due to environmental stress.
Of course, those are just guesses.  Does anyone know of any research papers in the area?

What is your reasoning behind your statement "some of this DNA damage is due to the DNA being transcribed heavily, or in an open chromatin conformation more often"? Just about every cell type has its portion of genes that are being heavily transcribed, even if it's just "housekeeping" genes like actin and GAPDH.

That's a good point about actin and GAPDH.  My understanding is that tightly packed heterochromatin is  more "stable" than euchromatin because it is energetically favorable to remain wrapped around histones.  Additionally, the conformation induced by the DNA/histone coil prevents many of the nucleotides in heterochromatin from being exposed to the nucleoplasm, which would decrease its exposure to DNA damage agents compared to uncoiled euchromatin.

There seems to be some solid evidence that transcription promotes mutation because the untranscribed strand is able to form secondary structures which expose bases to chemical mutagenesis.
Here is a recent paper about transcription-associated mutagenesis:
Kim H et al.(2010) Transcription-associated mutagenesis increases protein sequence diversity more effectively than does random mutagenesis in Escherichia coli. PLoS One 5(5):e10567. doi: 10.1371/journal.pone.0010567.
From the abstract:

During transcription, the nontranscribed DNA strand becomes single-stranded DNA (ssDNA), which can form secondary structures. Unpaired bases in the ssDNA are less protected from mutagens and hence experience more mutations than do paired bases. These mutations are called transcription-associated mutations. Transcription-associated mutagenesis is increased under stress and depends on the DNA sequence. Therefore, selection might significantly influence protein-coding sequences in terms of the transcription-associated mutability per transcription event under stress to improve the survival of Escherichia coli.

The authors cite a number of papers in their introduction which document the phenomenon that you could follow up. Just in case the focus on a bacterial system puts you off, the Kim et al. paper has in turn been cited in:
Wright et al. (2011) The roles of transcription and genotoxins underlying p53 mutagenesis in vivo. CARCINOGENESIS 32:1559-1567
Abstract in full:

Transcription drives supercoiling which forms and stabilizes single-stranded (ss) DNA secondary structures with loops exposing G and C bases that are intrinsically mutable and vulnerable to non-enzymatic hydrolytic reactions. Since many studies in prokaryotes have shown direct correlations between the frequencies of transcription and mutation, we conducted in silico analyses using the computer program, mfg, which simulates transcription and predicts the location of known mutable bases in loops of high-stability secondary structures. Mfg analyses of the p53 tumor suppressor gene predicted the location of mutable bases and mutation frequencies correlated with the extent to which these mutable bases were exposed in secondary structures. In vitro analyses have now confirmed that the 12 most mutable bases in p53 are in fact located in predicted ssDNA loops of these structures. Data show that genotoxins have two independent effects on mutagenesis and the incidence of cancer: Firstly, they activate p53 transcription, which increases the number of exposed mutable bases and also increases mutation frequency. Secondly, genotoxins increase the frequency of G-to-T transversions resulting in a decrease in G-to-A and C mutations. This precise compensatory shift in the 'fate' of G mutations has no impact on mutation frequency. Moreover, it is consistent with our proposed mechanism of mutagenesis in which the frequency of G exposure in ssDNA via transcription is rate limiting for mutation frequency in vivo.


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How can chromatin state be measured?
I have some RNA-Seq data and I'd like to align it to the physical genome and see which sections of chromatin are geometrically open and being transcribed.  The data are already sequence-aligned, and every Google search I do for tools that might help ends up sending me down a rabbit hole.  What computational tools have helped you?

Starting out with RNA data is great, since you already have fully spliced entities, despite  being in a different dynamic regime.
As the chromatin landscape itself is dynamic and High throughput data exploration has only begun in the last decade, consider the following tools and results with care...
You may find the following tools helpful:

Archalign , Publication (didn't work well during my day of trials)
Ensembl chromatin-state map data (See the database layout, Regulatory Build, and the lincRNA
genes in the web-interface of the Ensembl databases)
HaploReg, Publication (the most recent)


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How does "inheritance of methylation" of DNA and/or histones work?
What are the current models/ideas describing the mechanisms explaining inheritance of methylation on DNA resp. histone level?
Is there evidance of this "setup" information being really duplicated/inherited during a cell division process?
Pointers? Articles? What is the current state of knowledge regarding this?

Jon Wilkins has a nice introduction to imprinting.  He does a nice job of introducing the idea methylation and how these patterns are maintained during development and cell division.  Further, he links to some interesting papers on the subject.


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Which leads are abbreviated typically as channel ECG 1 and ECG 2?
I want make sure about the fact in the documentation of MIT-BIH NSRDB here.
They just say ECG 1 and ECG 2 but not what particularly.
I think they are the frontal plane channel and transverse plane channel but in which order, I do not know.
In their other database (MIT-BIH Arr.), #1 Channel was about MLII, but #2 Channel about V1.
So I am confused.
Normally, I get data which says leads are this and this. Display channels can be this and this. Etc with this device, very clear manual.
Ticket: https://github.com/MIT-LCP/physionet/issues/21

I'm voting to close this question as off-topic because it is about electrical schematics, not biology. Please take the [tour] and carefully read through the [help] to learn more about the site, including what is on-topic and what is not, and how to ask a good question.

Can you just ask them?

Answer in the ticket but it is still little open because it does say which lead is ECG1 and which ECG2

These records are from Holter recorders and the leads are not in
  standard precordial positions. As a general rule one lead is
  approximately a V5 position or a modified lead II (recording from
  right pectoral area to the cardiac apex), and the other lead is
  generally close to a V2 position.


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Regulation of chromatin structure
Recently, I reviewed the different levels of chromatin structure. The primary level is nucleosomes, where DNA is bound to histones, and has structural similarity to "beads on a string." The secondary level is a 30nm fiber, and the tertiary level is formed by radially looping the fibers.
I've also been learning about the histone code and how different modifications to the core histones relate to transcriptional regulation. Are these modifications the primary regulation mechanism for chromatin structure? In other words, does chromatin assume the most compact structure possible until histone modifications are made to enable transcription? Or have other regulatory mechanisms unrelated to transcription been discovered and characterized?

AFAIK chromatin would not assume the most compact structure without topoisomerase activity.

"Are these modifications the primary regulation mechanism for chromatin structure?"
It depends on how you define primary, we might currently think of histone modifications as primary because other regulatory mechanisms have not yet been well studied. Something else you can think of are the various regulatory proteins that interact with histone marks to modify chromatin.
I don't think you should imagine chromatin as assuming "the most compact structure possible until histone modifications are made to enable transcription" but rather histone modifications being a dynamic process with various transcription factors (a class of proteins) coming in and adding/removing histone marks as well as 'remodeling chromatin' (adding/removing nucleosomes)
As a sidenote, I don't believe much is known about the tertiary stage you initially mentioned so maybe that could play a huge role in the regulatory mechanisms of chromatin structure, it has just not been well explored.


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ChIP-seq and the output of SPP
I have been processing some ChIP-seq data with the R package spp. I looked through the literature (ENCODE uses it) and it seemed that spp is indicated as a good program to use.  I have found and adapted the two tutorials I found for using spp (first and second), and read the original paper. I have also e-mailed Prof. Karchencko and posted on the bioconductor listserv - all with no response. My question is about the MSER and the predicted sequencing depth - additional output of spp. So what I think the MSER is, is the score value above which a peak is authoritatively determined? When I look at peaks above this score value they are very well defined. Also, is the predicted sequencing depth is a prediction of the number of additional "tags" necessary so that the MSER and the FDR value coincide? It is hard to find decent information about this program.  Any advice or additional information is greatly appreciated!
TIA

does the inline spp documentation(> ??spp) explain it any better?

I found the answer in this paper, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3191340/

especially this figure,http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3191340/figure/F3/

There are no inline spp docs.

if you type in ??spp at the R prompt you'll get the docs. They're not expansive, but they explain the different parts of the library.

If you've found the answer, perhaps you might consider posting it as a full solution in the answers section so that others with the same difficulty can see it?

I found the answer to this question and some more interesting details that people who stumble upon this might also need. First look at this paper, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3191340/ and especially this figure, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3191340/figure/F3/. The MSER is a ratio of the fraction of discovered peaks to the fraction of the sampled reads to recapitulate them, so if you are able to return all of the peaks with a small sample of reads then you have excellent coverage. Now even though you have great and well defined peaks you need another sample to confirm that they are indeed significant, and you can do this with the IDR, see this website - https://sites.google.com/site/anshulkundaje/projects/idr. The IDR is laid out pretty well in the supplemental material for this ENCODE paper, http://www.nature.com/nature/journal/v489/n7414/full/nature11245.html. Beautiful!!


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How long can I store extracted RNA?
If I extract RNA from a (leaf tissue) sample using a one-step phenol:chloroform extraction, how long can those samples be stored at -80°C? And how many times can I defrost and refreeze them before they will become degraded?

I'd guess that this will depend heavily on the purification of the sample, pure RNA (no Mg2+ e.g) can be stored for a very long time at lower temperatures. Pure RNA in water can be stable for months at 4°C. Freezing and thawing RNA can affect the folding (for RNA with important tertiary structure) as I have observed.

If you're worried about freeze/thaw cycling, aliquot them into multiple tubes -- that way you only thaw what you need to use.

We can keep extracted RNA in -80°C for a few weeks, but before the start of any experiments, it needs to be validated by gel electrophoresis.

I keep my RNA in 1mM sodium citrate pH 6.4. Citrate is a chelator, and helps trap the divalent metals many RNAses need to work. The lower pH also helps inhibit RNAse activity. EDTA could work as a chelator, but it only chelates well at pH's where RNAse activity is worse. Citrate gives both chelation and low pH.
Of course I also keep my RNA at -80.

Do you need to do anything special to remove the citrate when it comes time to use the RNA, for example to make cDNA?

I've never noticed any problems, but I've never really checked. Only been working with RNA for a couple months now, and my lab is in an old building with a lot of dust, so RNAse contamination has been a bigger issue than enzyme activity. An isopropanol precipitation should be enough to clean up the RNA before any reactions if citrate proves to be a problem.

We're also in an old dusty building, and RNase is a problem for us too. Thanks for the tip - I'll check whether it works for RNAseq purposes.


text:
Can any protein be phosphorylated?
I am working with an Arabidopsis mutant with an F-box protein knocked out. It has been shown that F-box proteins targets must first be phosphorylated (Skowrya et al., 1997). I have heard of phosphorylation sites, but I can't find out whether every protein has them. Can any protein be phosphorylated?

Skowyra, D., Craig, K.L., Tyers, M., Elledge, S.J. & Harper, J.W. (1997) F-Box Proteins Are Receptors that Recruit Phosphorylated Substrates to the SCF Ubiquitin-Ligase Complex. Cell. 91 (2), 209–219.

One important thing is missing in the other answers: not only phosphorylation will happen only at selected aminoacids, but it will not happen at all of those.
So, not all of the Ser/Thr/Tyr of a protein can be phosphorylated because they could be structurally unaccessible to protein kinase and because they need to be in a specific motif in order to be phosphorylated.
The Human Protein Reference Database, for instance, lists the phosphorylation motifs for many Tyr and Ser/Thr kinases.

@leonardo: you did not speak about phosphorylation motifs which are a requirements for phosphorylation. You don't need just a Ser, you need a Ser with specific aminoacids around it.

@leonardo: no problem, I re-added the conformation problem just for completeness :)

Phosphorylation requires exposed serine, threonine, tyrosine, or histidine residues (in eukaryotes).  This is because the transfer of phosphate groups to proteins is mediated by a class of proteins called kinases.  Kinases can have broad or specific activity.
This review ought to have most of the answers to your questions :
http://www.cell.com/abstract/S0092-8674(06)01274-8

I like Nico's response best +1. I did find an interesting review on phosphoarginine and phospholysine - the list of possible phosphorylation sites grows. Not only is it the spatial context of the amino acid to be (or not) phosphorylated, as Nico writes and Leonardo implies, but also the temporal. Are the target protein and protein kinase co-expressed? If you're looking at specific tissues (root, flower, leaf) and the kinase in question is only produced in seedlings, maybe that potential phosphorylation won't really occur.

For one of the most comprehensive databases of protein post-translational modification (including phosphorylation, methylation, acetylation, ubiquitination, etc.), check out PhosphoSite. You can find links to sequences, diseases, motifs, publications, antibodies, mass spec experiments, structures, you name it.


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How do I clean and calibrate pipettes, and how often should I do it?
I work in a lab where all the pipettes are shared. We often have visiting students who come and use the pipettes for a short project. So when I work with them, they might have been handled by other people several times since I last used them. This leads me to worry that they might have been mishandled, or become less accurate somehow.
How do I clean and calibrate pipettes? And how often do I need to do it?

How often you should calibrate your pipettes depends on the tolerances of your application. For some applications, like quantitative PCR setup, one may care a great deal; for general lab work, one can probably be less particular. You can get a sense of how accurate your pipettes are by pipetting DI water onto the platform of a sensitive (and calibrated!) balance. Comparing the random and systemic error you get while doing that over several trials to the specifications published by the manufacturer will give you a good idea of whether pipette calibration will help and help give you a sense of how much you care. Note also that the error may vary with the volume; you should test error over the entire volume range you intend to use the pipette for.
How to clean and calibrate pipettes is specific to each design. Some pipettes, like Gilson's, are not intended to be calibrated by the user. Others are. The pipette user guide published by the manufacturer should tell you.
For some demanding applications, even well-calibrated and well-handled micropipettes may not be sufficiently accurate. In particular, I've made standard curves by serial 10-fold dilution for quantitative PCR both a) relying on my micropipettors to be correct and b) trusting pipettes only to get me into the right ballpark, and intentionally undershooting and correcting volumes with sequentially smaller pipettes until the reading on the balance is correct, if that's at all clear. In both cases I was using 900 ul of diluent and 100 ul of the previous dilution, which I feel are comfortably large volumes to handle. I found that the latter curve gave me noticeably better regressions.

Just a small note that hasn't been mentioned elsewhere.  Pipettes remain in calibration longer when reset to their maximal volume when not in use.  This is because it allows the spring (or other compression device) to relax fully and not be forced to stay in a compressed position.  This is an oversight I see in labs all the time, and something I always add to SOP's.

When doing a recent investigation into methods of quantitatively determining the concentration of aspirin in solution I found these procedures helpful.  They are a rough guide; design and procedure will vary by model.  It may be wise to check the manual that came with it, however in summary:
Calibration

Performed at least once per week
Performed in a room of the temperature indicated on the glassware (or at the very least in a room where the temperature will not fluctuate during the calibration).

For example a pipette I was using was a 10ml graduated, the procedure for which was:

Rinse the 10 mL pipette with 10 mL of water. Pipette 2 mL into a tared vial containing water on a 2-place balance. Document the weight. Set the pipette to 5 mL, pipette 5 mL into the vial and document the weight. Set the pipette to 10 mL, pipette 10 mL into the vial and document the weight.

However procedures are given in the linked document for pipettes down to 100μL.   Then compare the values from the mass with the acceptable values in the appendix.  A calibrated pipette will give ±1% of the mass indicated in the appendix, for example the pipette I described above:

1.98-2.02 g
4.95-5.05 g
9.90-10.1 g

Cleaning can be achieved by pipetting up and rinsing with distilled water (in my case) or more likely for your case 'nanopure' water.


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ChIP-seq vs ChIP-exo
I'm currently investigating ChIP-seq vs. ChIP-exo for finding binding sites.
As far as I can tell, ChIP-exo seems to be better in every way than ChIP-seq... but then again, I'm not strong in this area.
Are there any weaknesses in the ChIP-exo approach?

just as a first pass I'd say the extra ligation and primer addition steps for CHP-exo might resolve fewer hits in a given run, though that might also make your results less noisy as well.

ChIP-exo does seem to be the "ChIP-seq killer." I've seen Dr. Pugh present it a few times, and the audience is pretty much always impressed.
One thing I'd do if I were of the "experimental bent" would be to add random degenerate barcodes in the library prep to control for potential PCR artifacts. I imagine that since the "peaks" in ChIP-exo seem to be quite a bit narrower than ChIP-seq, I reckon it'd harder to use some coverage-in-local-neighborhood to remove such biases (assuming you wanted to do so).
To give you an idea of what I'm thinking of -- it would be somehow similar to what is depicted here.


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Why is HIV so large compared to other viruses?
The HIV-1 virus is about 120-150 nm in size and has a genome only about 10,000 base pairs long.
Other viruses are far more efficient, for example lambda phage has something like a tenth the volume and a genome five times longer. In other words, while half the volume of a lambda phage is taken up with DNA, in HIV it's more like 1%. Why is HIV so space inefficient?

One point is that lamba infects bacteria, which are more simple than eukaryotic cells.

There are many other things inside the HIV capsid besides RNA. The ssRNA is bound to the p7 nucleocapsid protein, the p6 late assembly protein, as well as integrase and reverse transcriptase, both of which are vital for infectivity. Also enclosed within the virion are Vif (Viral Infectivity Factor), protease, Nef (Negative Regulatory Factor), Tat (HIV Transactivator), and Vpr (Viral Protein R). Surrounding the capsid is a matrix formed by the p17 viral protein, and finally the lipid membrane envelope (taken from the host cell upon budding), which contains host cell proteins as well as the vital trimer of gp120/gp41 heterodimers, which bind to the target cell.

From Wikimedia
HIV (and many other viruses) depend on binding to certain cell surface receptors, and a larger total size increases the chances of binding to multiple receptors and potentially triggering receptor-mediated endocytosis, if that is part of the virus's entry strategy.
The naked λ phage binds to the lamB porin of single-celled prokaryotic E. coli by way of the phage's J protein and injects its DNA load into the bacterium, where host factors circularize and supercoil it, allowing for transcription to begin. While the phage's genomic material can be integrated into the host's chromosome, it is not required. Additionally, there is no nuclear barrier to cross.

From Wikimedia, numbers in parentheses are copies per virion.
On the other hand, HIV is a highly complex enveloped lentivirus that targets very specific cells in its multicellular eukaryotic host HIV requires reverse transcription and the assembly of a large pre-integration complex to transport the viral DNA into the nucleus and integrate it into the host's genome, all before it can even start producing new viral proteins.
Trying to compare the "packing efficiency" of HIV to the λ phage isn't just comparing apples to oranges, it's comparing apples to goats. The two viruses are completely different, with the only essential similarities being that they are viruses that infect cells.

First, your idea that "efficiency" has some relation to "genome size" divided by "diameter" is odd. You end up with arbitrary numbers that are not any kind of measure of "efficiency".
Second, HIV is an enveloped virus, while lambda phage is not; the internal viral core, less than half the size, is a better comparison.  Other enveloped viruses like influenza (100 nm, 14000 bp) are in the same ballpark.  Even many non-enveloped viruses, like Tobacco mosaic virus (40x300 nm, 6400 bp) have similar or "worse" ratios than HIV.
But the most important point is that it doesn't much matter.  It's not a measure of any kind of "efficiency" in the biological sense.

First, that's not my idea. My idea is that "efficiency" could be related to cell volume dividing by genome volume. That's called "packing efficiency". If you think DNA packaging is weird and biologically meaningless, you're simply wrong. See any number of references that immediately come up when searching for DNA packaging

@MarkE can you clarify what you mean? Everything that comes up for me when searching for "DNA packaging" has to do with chromosomes and how that interacts with protein production - but viruses by themselves don't produce any proteins in that state.

Obviously DNA packaging is important.  But obviously your measure of "efficiency" is meaningless.  Why?  Because so many viruses violate it.  You've created a hypothesis ("DNA:Volume ratio is important"), facts don't match it (phage to HIV have a huge range), so your hypothesis is wrong.  Facts always beat hypotheses.

I created no such hypothesis. You read the word "efficiency" and put meaning into it yourself that I did not imply. Regardless, see "Cell Biology By the Numbers", pp 9 "the viruses show strong economy of size, minimizing the required volume to carry their genetic material". If some viruses do show this economy of size and some do not, there must be reasons for that based on what the viruses do. Figuring out what those reasons are would be an excellent answer to the question.


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Are dark skinned natives of Central America dark skinned due to the same genes as people of African heritage?
I'm trying to understand how strong the evolutionary pressure on genes for skin color happens to be. Was it fast enough that the humans that migrated through the Bering Strait had lighter skin color and evolved darker skin color again afterwards?
Do the genes that give natives living near the equator in central America exist in African populations as well or are they new genes, that were likely evolved in that Central America?

The equator actually runs through South America, not Central America, so you might say tropical regions, rather than equatorial.  Also, you need to specify whether you mean the current inhabitants of the region, or the pre-Columbian ones.  I would suspect that there has been quite a bit of admixture of African (and European) genes in the current population.

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


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What are the visual, behavioral similarities and differences between Yellow Fever Mosquito (Aedes aegypti) and the Tiger Mosquito (Aedes albopictus)?
The Tiger and Yellow Fever Mosquito visually look very similar.
Recognizing the difference is important for tracking and controlling the spread of Yellow Fever, Dengue fever and other diseases. Especially important, now that the range of the two have overlapped in the United States.
I have some questions about the commonalities of these mosquitoes.

Evolutionarily, How can the similarities be explained?
Why do they both have the black body with white stripes?
What is the best way to tell them apart?

A. aegypti, the Yellow Fever Mosquito

A. albopictus, the Tiger Mosquito

Assuming that Aedes is monophyletic, the similarities between them can be explained simply by the fact that they share a very recent common ancestor and are therefore very closely related!

For the best way to tell them apart, see this guide: http://www.cdc.gov/dengue/resources/30Jan2012/comparisondenguevectors.pdf
Specifically, it seems that the silver pattern on the torso is different (lyre-shaped for Yellow Fever Mosquito, more linear for Asian Tiger Mosquito).  Location is probably also an important determinant.

+1; thoracic markings are the way to do it. I'd recommend adding a photo or image comparing the two (e.g. http://www.nzdl.org/gsdl/collect/who/archives/HASH017f.dir/p010a.gif) to your answer..


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

In plants, dsRNAs targeted to CpG islands within a promoter can also
  induce RNA-directed DNA methylation3, 4, 5, 6, 7, 8; however, it
  remains unclear whether gene silencing mediated by DNA methylation can
  be induced by dsRNAs in mammalian cells. Here, we demonstrate that
  short interfering RNAs (siRNAs; 21–25-nucleotide RNA molecules) induce
  DNA methylation and histone H3 methylation in human cells.

This paper seemed to show that siRNA can also induce long-term silencing of genes by DNA methylation in mammalian cells, which hasn't previously been observed. Unfortunately, the paper was retracted later, calling the results into question.
I found the paper very exciting at the time, as it suggested the possibility to perform gene therapy, to silence specficic genes, just by using RNA interference.
What is the current scientific consensus here, is there any convincing evidence that siRNA-induced DNA methylation is possible in mammalian cells? Was anyone able to replicate the results from Kawasaki and Taira?

From a brief survey of the literature, it seems Kawasaki and Taira have been largely vindicated by the community before and since their paper. The retraction was by Taira alone, Kawasaki refused to co-sign because he maintained the data were valid. From the retraction it seems the reason for the retraction was a lost lab book.
Prior to the Kawasaki and Taiga paper, there were already several other papers describing RNAi-induced epigenetic modification in mammals:

Castanotto, D., Tommasi, S., Li, M., Li, H., Yanow, S., Pfeifer, G.P. & Rossi, J.J. (2005) Short hairpin RNA-directed cytosine (CpG) methylation of the RASSF1A gene promoter in HeLa cells. Mol Ther. 12 (1), 179–183.
Morris, K.V., Chan, S.W.-L., Jacobsen, S.E. & Looney, D.J. (2004) Small Interfering RNA-Induced Transcriptional Gene Silencing in Human Cells. Science. [Online] 305 (5688), 1289 –1292.

Then there was another paper the next year by Kawasaki & Taira, which was not retracted:

Kawasaki, H. & Taira, K. (2005) siRNA Induced Transcriptional Gene Silencing in Mammalian Cells. Cell Cycle. [Online] 4 (3), 442–448.

And it seems the idea is now generally accepted and supported by quite a few other studies, mainly looking at germline cells. For example:

Carmell, M.A., Girard, A., Kant, H.J.G. van de, Bourc’his, D., Bestor, T.H., Rooij, D.G. de & Hannon, G.J. (2007) MIWI2 Is Essential for Spermatogenesis and Repression of Transposons in the Mouse Male Germline. Developmental Cell. [Online] 12 (4), 503–514.

There is no review that I could find which really ties it all together convincingly, but there are several reviews on RNAi directed histone and chromatin modification in general which mention that the fact is now established in mammals:

Joshua-Tor, L. & Hannon, G.J. (2010) Ancestral Roles of Small RNAs: An Ago-Centric Perspective. Cold Spring Harbor Perspectives in Biology.
Volpe, T. & Martienssen, R.A. (2011) RNA Interference and Heterochromatin Assembly. Cold Spring Harbor Perspectives in Biology. 3 (9).

The germ cell reference (Carmell et al., 2007) does not describe siRNA, instead it deals with entirely different class of small RNAs, called piRNA (in the paper they referred to them as rasiRNA, but piRNA now is a commonly used nomenclature). There are crucial differences between siRNA and piRNA (size distributions and proteins with which they associate) and they shouldn't be mixed up.

I browsed through Joshua-Tor and Hannon (2010) review that you cited and I haven't noticed any mentioning of siRNAs directing DNA methylation (or histone modification) in mammals. Sorry if I missed it, can you please quote the relevant sentence?

@Sergei you're right! That paper actually mentions piRNA-directed methylation, rather than siRNA. I'll leave it in the answer as it is still of relevance to the question.


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Does GTP-γS (GTP gamma S) bind all GTP-binding proteins?
I've just read an article Rab10 GTPase regulates ER dynamics and morphology - Nature Cell Biology 15, 169–178 (2013) doi:10.1038/ncb2647. In this paper, to identify Rab proteins in ER, first they isolated ER vesicles. Then washed them to remove cytosolic proteins. After washing, these vesicles were solubilized by detergent. Then, it was loaded on to a GTP-agarose column (to bind GTP binding proteins to the column). In the last step GTP binding proteins were eluted with GTP and analysed by SDS-PAGE.
As a control (before loading on to the column) they used GTP gamma S, because it prevents GTP-binding proteins from binding onto the column.
When I looked at their figure; on the control lane, there are lots of bands! So I'm confused :(If it prevents GTP-binding proteins from binding onto the column, after eluting from GTP agarose column why there are bands? And what can these bands be?)

If you can add a link to the article (hopefully it's open-access) that would be helpful in interpreting the results.

The title of this article is ''Rab10 GTPase regulates ER dynamics and morphology''
Link: http://www.nature.com/ncb/journal/v15/n2/full/ncb2647.html
Unfortunately, it is not open-access..

I can still see the figures, though. Which one did you have the question about?

Sort of related: http://biology.stackexchange.com/questions/3258/why-is-gtp-gamma-s-non-hydrolyzable

I had read the answer to the question in this link. But I couldn't understand exactly. So, does GTP-gamma S bind only to the GTPases very strongly? Is this the reason?

This is the figure the question is about. On the right is the control experiment with GTP-γS, on the left without it:

The bands that are visible in both experiments are unspecific binding. If GTP-γS doesn't affect their presence, the mechanism by which they bind to the column can't be specific to the GTPase functionality.
The proteins the authors were after are those that are present in the experiment without GTP-γS, but are not eluted in the experiment with GTP-γS. Those are binding via an GTPase functionality to the GTP on the column. They are marked with arrow heads in the figure.
The general idea behind this control experiment is to distinguish specific binding from unspecific binding to the column. Being able to do that means that the authors had fewer proteins they needed to investigate further, saving them some work.


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Is it possible to do chip-seq on a specfic region?
Is it possible to do Chip-Seq on specific region of the genome. The idea is to enrich before the sequencing step to have more sensitivity.

Are you interested in doing this in vivo or in vitro (by in vitro I mean adding the factor to naked dna and then crosslinking)? In what species?

If the question here is to perform an assay that only sequences from a specific set of segments of the genome, you probably could, but it would be a lot of work and you'd need a good reason to want to go through that trouble.
Not sure how you would limit the nucleotides you get back to a specific region of the genome say.  The entire point of a CHP-Seq experiment is to get as unbiased a set of nucleotides read and so using specific primers designed against only a specific set of the genome would be hard.
Usually CHP experiments focus the readout they are getting by only focusing on a specific transcription factor for instance.  The number of variants of short binding sequences that usually come out will easily be read by a sequencing lane.  You'd have to have a strong reason not to take all the data genome wide.  Generally the results for many such pulldown sequences are pretty short and frequently can't be found uniquely on the genome, esp if there are lots of binding sites for the protein to be found.
I could speculate that you might synthesize labelled primers from random segments of a cut up library coming from a chromosomal segment you cloned somehow.  That again that seems like a lot of work to cut back on the data you get from the experiment without a big reason for doing this.
Using a CHP-CHP experiment that only has specific regions tiled can make do something like this - this makes the chip itself more economical, making the experiment more accessible.
Hope this helps...

One way to increase power (if that is your goal) is to limit your mapping step: you only seek to map to a subset of the genome, e.g. promoters or transcribed regions.
I'll post a link later, but the reference is Leuleu et al. 2010 doi: 10.1093/bfgp/elq022


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Why is the administration of exogenous Anti-D not harmful to the foetus?
Haemolytic disease of the newborn can result from Rhesus incompatibility in utero.  In this disease an Rh- mother becomes exposed to the antigens of an Rh+ foetus by fetomaternal haemorrhage causing her to mount an immune response against the D antigen.  In her second pregnancy, repeat exposure to the D antigen activates memory cells causing a large production of IgG Anti-D antibodies that cross the placenta adhering to foetal red blood cells, which are then destroyed.
To prevent this, Rh- mothers are normally given exogenous Anti-D at 28 and 34 weeks gestation or after any sensitising event.  The aim of this treatment as I understand it is to 'mop up' any D antigen in the maternal bloodstream, covering it in exogenous antigen so the mother's immune system does not mount its own response.
I had previously assumed that these exogenous antibodies would be IgM to avoid them crossing the placenta, however looking at the summaries of product characteristics for some of the common preparations I see that "at least 95%" of protein content other than albumin is IgG.  I then wondered if the exogenous antibodies had a much reduced half life, however the half life is "about 3-4 weeks."
Why does the exogenous IgG not cause haemolytic disease of the newborn in the exact same way that endogenous IgG would in a previously exposed woman who had not had prophylaxis?  Is it dose-dependent or am I missing something entirely?

I've had a difficult time getting anything definitive to support my answer to your specific question of safety, however, I thought I'd go out on a limb and give you my understanding. A recent review of what is known about this can be found at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2809506/
I think you are on the right track with your 'dose-dependence' hypothesis. This is exactly what the makers of RhoGAM suggest, saying that the amount of antibody administered at 28 weeks into the pregnancy is sufficient to block any Rh antigen on Red Blood Cells that escape into the mother's blood, but insufficient to cross the placenta and be a problem. At this time the child will be large enough that any RhoGAM that does cross the placenta will not kill enough Red Blood Cells to harm the child.
Mom gets another dose of antibody after the delivery in order to ensure thorough coverage of antigen that results from mixing of blood at that time, however this dose is not a problem as the baby has already left the mother.
By the way, the rationale for the antenatal dose is that post-partum administration alone failed to prevent sero-conversion in almost 2% of Rh- women, putatively due to transplacental hemorrhaging.

That's an excellent article, thank you.  I had wondered if they used primarily IgG2 to reduce placental transmission but this is not the case (the article indicating IgG1 and IgG3).  If a 125 unit dose can neutralise only 1.25 ml of D+ve blood and not all of it crosses the placenta then this is likely to be an insignificant dose to cause harm.  I assume this could cause harm with a greater number of repeated doses after sensitising events but I think this is sufficient evidence to say that it is indeed a dose dependent phenomenon.  Thanks!

I think this just isn't really the kind of thing that can be explored in a dose escalation trial (at least not in humans) and it would be meaningless to try to extrapolate from any other species.

From the most up-to-date article I can find on the D-GAM treatment, IgG1 is primarily used, followed by IgG2. To be precise:
64.3% IgG1, 29.0% IgG2, 6.2% IgG3, 0.5% IgG4

Another treatment for this is Rhophylac. In this case, we have: 84.1% IgG1, 7.6% IgG2, 9.1% IgG3, 1.0% IgG4

Where you say "insufficient to cross the placenta and be a problem" - the review paper you link to actually makes this clearer. It explains that some of it can cross the placenta, but not enough to endanger the newborn.


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What happens to bones that do not heal?
I would like to know the steps the body takes to heal a broken bone and what happens if it cannot heal properly.  I am not looking for advice or explanations on resting and not doing activity, I am genuinely interested in understanding the outcome if activity is not stopped
Here is the scenario:
A rock climber develops a pain in the lower part of their middle finger.  The pain is not so bad so the climber continues to climb for four weeks, but the pain has not gone away.  The climber decides to go to the doctor fearing they have an A2 Pulley tear.
To their surprise the Doctors tests show no A2 pulley tear but rather a minor stress fracture of the Proximal Phalanx.  The doctor says stop climbing, however the climber has several trips planned and does not experience any pain while climbing nor a loss of strength or power while climbing.  The only time pain is felt is when the lower part of the middle finger is squeezed.  Because of this the climber decides to continue climbing as they just love it so much.
Question:  What will happen to a stress fracture in the finger if adequate rest is not given?
I understand the first thing the body does is try to stabilize the break and then starts to repair.  What happens if it is continually used and not given the chance to properly heal?  Will the climber just have a sore spot on their finger for the rest of their life or will it progressively get worse and worse?

Fracture healing occurrs in several steps:

haemorrhage: blood and surrounding cells fill the space created by the fracture.
fibrous callus: chondrocytes colonize the fracture space, with neovascularization.
bony callus: osteocytes colonize the fracture space and rearrange in woven bone
remodelling: in long bones, woven bone is remodelled into lamellar bone and creates a new haversian system

If the fracture is not immobilized enough (not enough rest), several things can happen that are mutually exclusive:

normal healing: luckily, the bone is not too much mobilized and is able to heal normally
fracture displacement: the bone heals, but in an incorrect alignment. This gets partially corrected over time, but the correction mechanism is limited, especially in adults.
pseudarthrosis: the worst case. instead of normal healing, cartilage grows to protect the bony extremities that are mobilized and prevents further healing. This causes pain, and will require surgery in most cases.

Exactly the answer I was looking for, well done. Thanks :)

Can non Union or delayed Union occur in such a bone?

@CRags non union of a phalanx? Yes, of course it can happen as in any long bone fracture. However, it is true that my answer refers more to an acute event than to a stress fracture, which would most likely cause more of a periosteal reaction/chronic pain & inflammation than displacement or pseudarthrosis.


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Plant anatomy, what are these stem like filaments growing under the flower
The picture below shows what I am talking about. Each flower has one and I am just wondering what they are?

Looking at this, it looks like a spur. A quite overbreed one, though.

These are "floral spurs" – they usually contain nectar, and are part of the variety of complex flower shapes that orchids and other flowers, like columbines, have co-evolved with their pollinators.
Essentially, in order for the pollinator to reach the nectar down in the base of the spur, it must move into a position where pollen is deposited onto it; then when it moves to another flower, it can transfer that pollen and the plant is able to reproduce.
Of course, long tongues are good at getting nectar from long-spurred flowers, so there is both species filtering for long-tongued species, and presumably also selective pressure within a species for long-tongued individuals. This, in turn, drives selective pressure on the spurs.
Spurs are a classic example of a "key innovation": they have evolved separately in different types of flowers, and when they evolve, they often lead to rapid speciation (because a small change in spur architecture can constitute a barrier to reproduction). (See, for example, publications of Scott Hodges)


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RNA seq and using of Poly(A) or non-Poly(A) based amplification of RNA
I'm studying "Deep sequencing the circadian and diurnal transcriptome of Drosophila brain" Hughes et al., 2012. I've got some problems with the materials and methods.
Before RNAseq, the authors amplify RNA. They use two kits: Poly(A) based and non-poly(A) based. For the samples Poly(A) based amplified they do a sequencing to generate 100 bp paired-end and for the samples Non poly(A) based they do a sequencing to generate 75 bp single-ends.
I read in the strategy:

Total RNA was amplified, and ribosomal RNA was depleted, using a non-poly(A)-based amplification kit, which significantly diminishes the 3' bias in downstream libraries used for RNA-seq (see Methods).

I don't understand why they use both kits. Actually, they say that they want to asses the bias introduced by an alternative amplification methods BUT the following sequencing is not the same in both cases so there is more than one parameter which change between the two protocols so I don't understand how they can compare.

For the poly(A) based I understand that the rRNA are not polyadenylated so they remove rRNA from the amplified sample. However, don't they remove non-coding RNA too (long or short)?

For the non poly(A) based, I don't understand how they remove the rRNA but I understand that since they don't select the RNA according to their poly(A) tails they keep the rest of the RNAs which are not kept in the poly(A) based amplification.

When I look to the RUM statistics, I see that there are fewer total aligned reads in non-poly(A) based: 70-80% (non polyA) and 80-90% (polyA). So it seems like we lose some information with non-poly(A) based.

To sum up, why the authors use Poly(A) and non-poly(A)? Why do they show all the rest of the results from the non-poly(A) amplification since they have less aligned read? How do they compare since the following sequencing is not the same? How the rRNA are removed in both techniques and especially in non-poly(A)?
I didn't find the protocols kit with exactly the same name but here are the links to the protocols which are closer (I think).
http://www.mscience.com.au/upload/pages/nugen/nugen_ov_rna_seq-brochure.pdf
http://excilone.com/client/document/ug-arcturusE%E2%80%9E%C2%A2-riboamp%C3%82-hs-plus-amplification-kit-user-guide_38.pdf

Yes, it was odd that they said that. The second protocol with the polyA based method had another purpose though, which was "examine the reproducibility of period-regulated genes in an independent genetic background". I would suspect this was its main purpose, and not so much to look at fragment bias, which has already been done in other papers. They didnt even publish this data in the main paper, they put it in the supplement.
Yes, this would remove any transcript without a polyA tail.
One way of purifying is by using oligos that are antisense specifically to rRNA. The oligos will be attached to some solid phase, on a column or a bead or something, and the rRNA will stick to it and everything that doesnt is what will remain in your sample
It is not safe to say that you lose information with polyA based kits. The polyA based technique had reads that were single end and only 75bp long. This is compared to the other protocol which was 100bp paired. Reads that are 75bp will align much more easily than 200 bp (100 x 2 since its paired). However, there will not be as much certainty in the alignments, as there is a greater chance it will align to multiple locations in the genome, or align to the wrong location. So yes they have more reads aligned but I would be hesitant to make any conclusions from this. And if you look at total numbers of reads, the non-polyA method aligned more. Again, I dont think the main purpose was to show that one method is better than another.

Basically the method you choose depends on your experiment and your samples. Random hexamers will give you non-coding RNA which is important in some experiments, and that is its main advantage. It will also give more uniform coverage across the gene (no 3' bias). Poly(A) selection will give you only mRNA, however you will miss any transcripts that are degraded or partially degraded. If you have a high quality sample or a large amount of starting material this is less of a concern. As mentioned before you end up missing parts of the 5' end on some transcripts with this method. If you have a low abundance transcript that you are trying to find, polyA selection is probably best, as it is more likely to be captured and you will capture the whole thing, rather than just getting bits and pieces of it with random hexamers. They both have advantages and disadvantages. Hopefully this helps you or someone make a decision.


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Histone marks mechanism
I am slightly confused about the mechanisms that makes histone modifications associate with gene expression.
That is, H3K36me3 is believed to be present in actively transcribed genes, H3K27me3 in repressed regions, etc. What is the mechanism that makes the presence of H3K36me3 to be in active regions, etc.? Is it causal or just an association?

First we must remember what a nucleosome is: a segment of DNA packed with proteins called histones. This is the initial step to turn the DNA a more compact structure. Citing the advantages of an extremely compact but flexible structure like chromosomes would make this answer a little longer (if you want, I can add them later), and to answer your question faster, we'd better keep moving on the discussion. Epigenetics and gene expression are so beautiful that they have many mechanisms to regulate or change gene expression, and you mention one, which is done by reversible covalent modifications on histones. Such modifications are executed by especific proteins, that can do methylation, acetylation, phosphorilation in some aminoacids of histones. When an aminoacid of a histone is linked with charged groups (e.g: methyl, acetyl) its charge is altered, and depending of the resulting charge (negative or positive), it can reduce or increase the afinity between the histone tail and the adjacent nucleosomes, and therefore, make the nucleosome more compact and less accessible or less compact and more accessible, respectively. But the main effect of these covalent modifications on histones is that the groups added to the aminoacids act like markers indicating for proteins involved in expression (like transcriptional factors and RNA polymerase) that some genes are switched on or switched off. The patterns of this modifications (which one repress or estimulate gene expression) is not completely understood yet.An example of how a certain region is chosen to be marked for expression or repression: Consider an organism that has received a repressed gene related to obesity from its parents and this organism is lazy, eats a lot, has a sedentary life. As a result of all this stimulation, the proteins responsible for altering the histones of the gene related to obesity can be activated or recruited by others proteins, and make covalent modifications in a way that the histones of that gene now are marked for it to be expressed. This organism can pass the new mark to the its children and they will have propensity to obesity due to the activated gene. (This final example was an experiment of epigenetics using rats, I saw a long time ago, so I can't remember the reference).And about if it's casual or not, the marks of regulation in a sequence of DNA can't be casual. For each one or two modifications of histones there is an enzyme that will only do its function if some event is already triggered or if it's triggered during the cell life depending on the environment the cell is exposed to. So millions of cells of a same tissue that have the same function would not have the same pattern of expression for the main genes related with that function by chance. Therefore, regulation of gene expression is very refined and histone marks are one of the many mechanisms that together dictate if a gene is repressed or expressed.


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Benefits of CLARITY?
What are the benefits of CLARITY over this technique that was published more than a year earlier?
Of course the second technique needs a fancier microscope that is likely more expensive and requires more technical ability. But CLARITY requires an objective with a very long working distance. I'm no expert in optics, but I'm pretty sure this results in a significant decrease in numerical aperture and therefore resolution.
It seems as though the other method is simply better than CLARITY, and that a lab serious about doing whole brain imaging could afford to invest in a setup similar to the one described. Yet this technique received none of hype that was bestowed on CLARITY. Am I missing something?

To start with, CLARITY is a whole-brain imaging technique , there is no need for sectioning which destroys connections and can cause the brain slices to warp as they are being cut. The costs for setting up CLARITY are much lower because all that is needed is chemicals to get the prep ready for imaging whereas in the Nat Methods paper you have to set up an automated slicing and imaging system,not an easy task by any means. Therefore an investigator is more likely to use CLARITY due to its easy of setting up and low start up costs. Also, as with any brain slice technique , you lose patterns of connectivity among neurons that are more than the slice distance apart(i.e in the next slice) and you don't look at the "whole picture". Thus due to the complexity,high start-up costs and specialized machinery STP is a poorer method than CLARITY for whole-brain imaging.As for resolution, I agree with you on principle that CLARITY has worse resolution(I looked through the paper but couldn't find exact figures) although you have to consider this in terms of all the trade-offs.

I don't think this is fair to the serial two-photon technique. For each Z-stack the sectioning occurs after the optical slice is recorded, so I don't entirely see how the warping is a big issue. Also, even using CLARITY you eventually need to take optical slices, so you don't really get a "whole picture." You get a z-stack that then has to be computationally reconstructed. The same problems with this reconstruction that you point out (loss of connectivity patterns) is a concern in CLARITY as well.

This is not quite true. You don't need to section the brain with CLARITY because the how point is you clear away all the fats so you image cells directly in their native configuration without disturbing them. When I said slices I mean physically cutting the tissue which you do for STP but not for CLARITY. Hopefully this clarifies my response.

You don't need to physically section the brain, but you still need to take optical sections for CLARITY. Unless there is a microscopy technique for creating 3D images that doesn't involve stacking a bunch of 2D images, of which I'm not aware.

Right, but optical sectioning does not disturb the tissue like slicing, nor does it destroy connections or cause swelling.


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Where do the phosphate units come from when EGF units dimerize?
Upon EGF binding, EGFR units dimerize and cross-phosphorylate. The phosphate groups are transfered to intracellular c-terminal tyrosine rich regions. Where do the phosphate units come from in this cross-reaction? Is it from bound GTP units?

In protein phosphorylation, the phosphate group transferred from the kinase to the substrate comes almost universally from ATP. However, there is evidence of some protein kinases using GTP as their phosphate source, although they are few and far between.

I see, so it's similar to the insulin receptor.

@TMOTTM yes, and just about every other kinase out there, with the exception of CamKII, which I mentioned, and possibly some others. You can pretty safely generalize and say that the phosphate group in protein phosphorylation reactions comes from ATP.

Ok. Except for the GTP used by G-proteins.


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Relieving pain by massage
How come the pain in our body, primarily due to excessive physical exercises, vanishes after having a massage? What is the mechanism?

It probably increases the blood flow and so it is easier to clear out lactic acid, which can cause muscle pain.

Short Answer
What you're referring to is called Delayed Onset Muscle Soreness (DOMS).
We don't really know what causes DOMS, but it's most likely caused by a cascade effect started my muscle injury. Also, we aren't sure if massages (or what type of massages) really help relieve the pain.

What is Delayed Onset Muscle Soreness (DOMS)?
Delayed onset muscle soreness (DOMS) is the sensation of muscular discomfort and pain during active contractions that occur 24–48 h after strenuous exercise. The initial symptoms are most evident at the muscle-tendon junction and thereafter spread throughout the entire muscle.(ref)
Physiopathology of DOMS
The mechanism is not completely understood and several theories have been proposed, namely: (ref)

lactic acid
muscle spasm
connective tissue damage
muscle damage
inflammation
enzyme efflux

The underlying cause is probably a conjunction of 2 or more of these theories. However, ultimately, the underlying transversal cause is muscle microtrauma. There are a few interesting findings that support this:

High-intensity eccentric contractions induce performance decrements and DOMS (ref),(ref)

Concentric exercises seems to cause no soreness (ref)

Eccentric contractions are high force and are associated with muscle damage (ref)

Creatinine, Potassium(K) (and sometimes Myoglobin) excretion (urine levels) and Creatine Kinase(CK), K and Neutrophil levels in blood are increased after intense exercise. This is also seen in acute rhabdomyolysis, a condition in which damaged skeletal muscle tissue breaks down. (Ref: Harrison's Principles of Internal Medicine, 17th Ed, Chapter 273: Acute Renal Failure)

The effects of massage on DOMS
A massage MAY indeed reduce pain in DOMS (ref), however, most studies' results are inconsistent and hindered by methodological flaws(ref) such as lack of control of the placebo effect or the subjectivity of pain.
A study by Smith et al suggests that sports massage will reduce DOMS when administered
2 hours after the termination of eccentric exercise. They also found lower CK levels, higher cortisol levels and a prolonged elevation of neutrophils in the blood of massaged groups (ref). This might mean lower muscle trauma and inflammation due to a reduced migration of neutrophils (to the muscle) and higher levels of serum cortisol.
The increment in the blood flow to the muscles during a massage might also be implicated in the process, by restoring oxygen and nutrients required to muscle remodeling. However, there is no clinical evidence of this fact and a study strongly rebutes this (ref)
Hyperstimulation of the nerve endings in the affected muscles might give a sense of pain relief, in a context of microtrauma. (ref)


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Chromatin shearing: what is it and what are the effects of high-sensitivity?
I am reading a paper  which discusses a complex (MSL-DCC) involved in dosage compensation of the drosophila X-chromosome. Descriptions of the complex's structure and function are given in the papers introduction.
To start with, could someone explain what is chromatin shearing? what is happening during the process?
Secondly, the authors then say:

"Our data suggest that the complex is particularly sensitive to shear
  forces on transcribed chromatin so that only the direct interactions
  on MSL-3 with nucleosomes are retained" - discussion section (subheading: interaction of the MSL-DCC with target gene bodies)

Could someone explain what is meant by 'particularly sensitive to shear forces'? In comparison to what? And what are the consequences of it being more sensitive?

Shear forces are experienced by all molecules, and molecular complexes when they are treated roughly. So, for example, if you have a solution of high molecular weight DNA it will be very viscous, but if you treat it roughly by pipetting up and down through a narrow aperture the DNA strands will suffer shear forces strong enough to break covalent bonds and the solution will become less viscous. Another example of the use of shear forces in biology is lysis of yeast cells using small glass beads. As the slurry of beads and cells is vortexes the cells experience intense shearing forces (forces tending to pull different parts of the cell in different directions) and they break open. They are not being physically ground by the beads.
In the specific case that you quote, you apparently have a multimolecular complex (MSL-DCC) held together by weak (non-covalent) interactions and interacting with the nucleosomes through a component called MSL-3. Without looking at the paper, it seems that this complex is prone to falling apart (during isolation?) as a result of shear forces strong enough to overcome the non-covalent binding interactions. Think of a helicopter with a man hanging on to the undercarriage with both hands. Then imagine a second man hanging on to the first man's boot lace with his thumb and finger. Who will fall off first due to the shearing forces experienced as they are buffeted by the wind?
The helicopter is the nucleosome, the first man is MSL-3 and the second man is the rest of the complex.
added later in response to MattDMo
looks from this that I was wrong - it's actually a French press and other homogenizers that generate shear forces. I don't think I made the glass bead story up, but I can't remember who told me. The BeadBeater people also refer to "cell cracking" not shear. Apologies everyone, I think the rest of the post still holds up, and thanks to MattDMo for the challenge.

Fantastic analogy with the helicopter, I used that in the Journal Club today - turns out I wasn't the only one not familiar with the term. Helpful answer thanks.

Glad it was useful - after posting I was a little nervous about how it would be received.

I didn't know that cells were disrupted by shearing forces and not by the beads themselves. Do you have any reference for that? (not that I don't believe you, I'd just like to learn about it...)


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Is beauty universal?
I don't know if this is off topic at all, but there are a lot of things that humans tend to consider beautiful which other animals in nature also seem attracted to. For example, bees pollinate the most vibrant flowers. Birds of Paradise appear to demonstrate a selective advantage to looking pretty. Peacocks flaunt their looks. Songbirds sing beautiful melodies. Butterflies are amazing. Some spider species have bright colors and perform cute dances. It seems like these examples demonstrate an evolutionary advantage not just for things that stick out from a crowd but also that look good to us humans.
So I guess my question is, is beauty universal? Are there aesthetic characteristics that are attractive to all these different kinds of animals, that would signal something good for others to see, and that different animals including humans would universally recognize as being nice?

There's plenty of things that have evolved that are considered ugly by humans. Cherry picking examples allows one to make virtually any argument seem reasonable. Voting to close as primarily opinion based because beauty is entirely subjective and individual so can't be reasonably discussed in an answer.

Fitness
Fitness is a trait which, when high is pretty much always attractive to potential mates in all species. However, fitness is definitely not an "aestetic characteristic".
Traits that are often correlated with fitness
There are some "aestetic characteristics" that are correlated to fitness in many species, such as body weight. Such trait are therefore often correlated with what individuals find appealing.
Body weight is a standard examples. Heavier individuals often have better competitive abilities, better foraging abilities and therefore have a higher fitness. As a consequence body weight is often used a s a proxy for fitness.
Relative body characteristics
Novel traits in a population can be evidence of parasitism and therefore would tend to be considered as non-attractive. However, counter to this intuition, it has been shown typically in birds having high sexual selection, that individuals having novel traits are more attractive.
Why is there a lack of generalization?
To build the intuition I would just say that given the gigantic phenotypic diversity that exist among all species that perform some level of mate choice, it is pretty clear that you will not find a simple morphological trait that will be attractive to all species.
Mating preferences evolves. Individuals that are attracted toward individuals that will yield to a high progeny (and low sexual conflict and low transmission of parasites) have higher fitness their preferences increase in frequency in the population. As a consequence, individuals like whatever is (should rather say 'was') favoured in the population. Now, in some population individuals are selected to be small, or to be big or any other things. This is the process of natural selection applied to mating preferences.

I believe what you're referring to as "beauty" is completely subjective.  You say Birds of Paradise look pretty; I think otherwise.  I happen to find California Condors extremely attractive!  ;)
Cultural studies in humans show that what females view as sexy or attractive in one culture can be completely different from another.  The only common denominator seems to be an attraction towards symmetry in faces.  This has been shown by several studies in humans, but I don't know if it has ever been repeated in animals of different species.
Also different species of bees or other pollinators are attracted to different colors and smells in flowers.  They can be very selective.  Some species have a close relationship with and will only visit flowers of a single species, like the Yucca moth for example.  This again proves that "beauty" is subjective and that nothing is universally attractive.  Flowers that are visited by many species are not seen as more "beautiful."  They simply smell better, or have more nectar, or are easier to access.  I don't think there's any such thing as "beauty" in nature.  It's just an artificial label we have created...
Hope this helps!  :)

I agree with what you said. But I guess what I meant was if there is a hint of anything that inherently looks or sounds a certain way that is beautiful. I mean we can do the same thing about art, some people will say abstract art is good and others say there must be classical rules that are followed. And I for one am wholly unconvinced that abstract and classical just come down to taste, but I fear I'm veering too far into the philosophical. But maybe you can think of my question as, does nature have any "classical rules" of art that cause an above-average attraction.

Maybe I would find your answer more compelling if you demonstrated that random variation mixed with symmetry would produce the same type of ugly, normal, and beautiful distribution as what nature produces, and that then it'd be reasonable to say that we just pick out the brightly colored nice looking animals as beautiful because we coincidentally find them as nice as members of those species do (despite the near lack of colorful things in our own species). Oh, and maybe you like how vultures look more than birds of paradise, but overall people Indeed like the way the colorful birds look more.

"Is beauty universal" is a question that is haunting the scientists, philosophers, psychologists and common people for a long time. Some of them consider beauty as a subjective feeling whereas a second group of people consider it universal. Both of them have reasons to believe.
So I would try to discuss the topic in an evidence based way rather than expressing my own opinion about it.
Reasons to believe beauty is subjective -
The subjective nature of beauty in human beings is very easy to acknowledge. Here are some common examples -

Culture - Surma and Mursi women in Ethiopia wearing large lip-plate (clay or wooden plates inserted in upper or lower lip) and long necked women in Myanmar are considered to be beautiful in their respective cultures. Some cultures prefer black skin whereas most western people prefer white skin color.
Gender - a good looking man is considered more handsome to a woman that to another man. Same is true for females also.
Individual - individual perception of beauty is most diverse among all of these. What seems beautiful to me may not be so to you (I think this doesn't need further elaboration).
Specific groups - a mother finds her children beautiful even if it has some deformity.
Other species - sometimes it becomes confusing to differentiate the perception of beauty with other feelings in human and other animals. I am taking the examples you provided "Peacocks flaunt their looks." - the colorful feathers looks beautiful to most human beings. But it is actually a method to attract sexual attention of peahens (Courtship dance). Sexual attraction is a different feeling from beauty.
If the peacock example feels confusing to you, just take another easy example - most of us feel attracted to chocolates, but we may not consider it beautiful...so they are two separate feelings, right !
We love flowers, they are beautiful. But if you give a flower to a goat to admire its beauty it would at once put the flower into its mouth not even bothering to give it a glance.
Some things we feel beautiful may no have developed for that purpose at all. Tigers and zebras have those beautiful branding on their body to help them hide in grass field with two completely different purpose (one is predator and the other is a prey)
Note that awe and beauty are not the same things.

Reasons to believe that beauty is universal -
 1. In some studies symmetrical facial feature was found to be more attractive even to little children
 2. A person with good attitude and personality seems more beautiful to all.
 3. Shape of some body parts e.g. chin-shape (prominent chin in male and rounded chin in female) and vertical forehead are things that most people consider to be a part of beauty.
 4. "It is hard to find a purely biological basis of beauty", when a cat looks at you, it becomes difficult to know whether it is expecting food or is fearful or just admiring your beauty !! Much of the feeling in animals are yet to discover and thus evidences are few till now on this aspect. So, most evidences and scientific studies that you find with universal beauty are actually on human species as a whole. Studies on body shapes are an example.
 5. But if we consider attraction between animals (other than that for mating purpose) to be an indicator of the feeling of beauty, then certain properties like risk taking behavior, fitness etc may be considered to be universally beautiful.
So, there is enough evidence to consider beauty as both subjective and universal feeling.
Feel free to criticize this answer in the comment section below...but please give evidence (examples) with it, so that it can be a collection of all different thoughts in this topic. Please write down any mistake in this answer as I have written it in a hurry !!
For further knowledge you may follow these two links -
http://www.bbc.com/future/story/20150622-the-myth-of-universal-beauty
https://www.psychologytoday.com/blog/homo-consumericus/201004/beauty-culture-specific-or-universally-defined
Thank you all.

Wow I think this is a really good answer actually. I would have maybe liked a little more discussion about if it's a coincidence that what birds find attractive is what humans think is beautiful, because it seems across a huge array of bird species that where there isn't too much pressure to blend in with grasses or something, the male's way of attracting a female is by looking or sounding in a way we often consider nice or if there really is some kind of "beauty rule" applying there.


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Does a walk after a meal help with digestion?
I am a bit confused with the notion that "walking after meals helps you in digestion ",
Some say that it helps, whereas others oppose it. Can someone come up with a valid explanation for this?

This seems like a job for Myth Busters :D

apparently walking helps in the movement of food into the stomach and improves digestion. Also helps in decreasing blood sugar after meals, which decreases cardiovascular risk and potential signal diabetes by helping muscles absorb glucose in the blood. Here is the link to a study done comparing the results of walking after food and after a drink. Also a study was conducted where when people with Type 2 diabetes took a 20 minute walk just 15 minutes after eating, their post-meal blood sugar levels were lower than if they had walked before dinner or not walked at all. Here is the link to another similar study.

Well it depends what you mean by helping digestion.
This answer in skeptics may shade some light:

a walk after my lunch [is] a healthy habit that helps with digestion.

Walking may be a healthy habit but it does not specifically assist digestion.

Digestion is a process which takes place in resting conditions. Exercise is characterised by a shift in blood flow away from the gastrointestinal (GI) tract towards the active muscle and the lungs.

From: Is the gut an athletic organ? Digestion, absorption, and exercise. [Sports Med. 1993] - PubMed - NCBI
So it's seems walking help sugar level (but this is independent to having eaten just before) but seems to not be beneficial to the digestion (in particular stomach contractions ― nutrient absorption will only start in general two hours after your meal, once the food leave the stomac)


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What type of force holds my body rigid & doesn't make it collapse?
My query is that as atoms & its molecules have inter & intra molecular forces,like that only what type of force keeps the cells bonded to each other & prevent it from collapsing ever since.

Adding on to what Martin said. If you imagine an atom to be the size of a football stadium, the nucleus would be the size of a fly on the fifty yard line. The vast majority of an atom is empty space.
In the 'classical' view, atoms are mostly empty space and the reason they feel solid is that if you try and push them, the electrons around your atoms bump into the electrons around the other atoms and because they're both negative they repel each other and push away. You feel this force as 'solid'. It's like pushing a really, really stiff spring or trying to force 2 magnets together, except it's electronic in origin.
In quantum physics, each particle has a wave function. Wave functions can only exist in certain forms, and when electrons are bound in an atom their waves spread up over a big volume (it's the only way mathematically to get a stable wave, ie an electron that exists. Since it exists, its wave function spreads out much further than the thing you classically think of as an electron).
Electrons have something called antisymmetric wave functions, which means they obey the Pauli exclusion principle:
http://en.wikipedia.org/wiki/Pauli_exclusion_principle
You can't have 2 electrons in the same state (and 'state' includes 'space'. You can have 2 oppositely spinning electrons, but no more in the same place). So atoms spread out because you can't squeeze the wave functions in. The rest of it is electrostatic repulsion just like in the classical model.
I'm sorry if that sounds a little complicated, but it's a combination of quantum physics (which doesn't really make sense, even though it works!) and electronic repulsion (from like-charges-repel).

Fundamentally, the most important physical force in everyday life and most cell-to-cell as well as molecule-to-molecule interactions are electromagnetic interactions. It is mainly electrical interactions that turn amino acids into intricate 3-dimensional proteins, as well get molecules attracted and specific for cell receptors.
The reality is somewhat more complex, as quantum mechanical (e.g. in charge distribution and fluctuations) effects can affect the attraction or repulsion of things temporarily.

Sir,i still have a doubt that from where these electromagnetic interactions come from.