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Maternal immunity without prenatal vaccination?
It is recognized that antibodies from the mother provide a level of protection to infants. This is why mothers are often advised to get vaccinated when they're pregnant. However, does the vaccine have to be given during pregnancy or would a vaccine taken as a child-teenager, if still effective as an adult, function the same way?
I'll try to give an example to clarify. Suppose a person is fully vaccinated against Whooping Cough, at least based on the current schedule, but does not receive a pre-natal vaccination. Is it reasonable to suppose that if the vaccine is still effective and is causing the body to produce antibodies, that these maternal antibodies would be passed on such that the infant would still receive some protection that it would not receive had the mother not received the childhood vaccinations?
Further Detail
I am interested in this question because it could influence studies which look at rates of infection/cases in infants, in order to measure the effectiveness of cocooning. These studies assume that because these children are too young to be vaccinated, the vaccine cannot be influencing their susceptibility, except through cocooning. But if childhood vaccination results in a high enough IgG levels, that might not be the case, and so this result should be considered in such studies as this one.

Pre-conception and prenatal immunizations are recommended for several reasons, protection of the mother (during a higher risk period), fetus, and newborn. Pertussis is a very particular case. Can you clarify whether you're asking for your own family, or as a matter of general interest?

This is an immunological question. To what degree, if any, does vaccination of a person when she's young, increase the odds of maternally derived immunity against the pathogen?

got it. let me know if there's anything i didn't answer for you!

tl;dr

If a woman was vaccinated when she was a child, will she be able to provide passive immunity to her children?

It depends on the vaccine. Generally, if the mother still has high levels of protective IgG  during the third trimester (especially if it's type 1 or 4), those antibodies will cross the placenta and provide passive immunity to the newborn. In whooping cough, though, good levels of newborn IgG require vaccination in the third trimester.
Maternal immunizations
Immunizations are given to adult women prior to conception, while pregnant, and after delivery to protect the mother from infection, protect the fetus from infection, protect the fetus from congenital anomalies associated with maternal infection, and to protect the newborn from infection through passive immunity and through decreased exposure to infected contacts. The exact recommendations differ for different vaccines and different levels of maternal risk. From the same recommendation, you can see that some vaccines are recommended only if maternal antibody titers are negative and some are recommended regardless of maternal antibody titers or prior immunization.
Passive immunity of the newborn
IgG undergoes active transport from the maternal circulation to the fetal circulation across the placenta.

The reference linked above is an excellent review of transplacental antibody transfer and the science that underpins maternal immunization recommendations for passive immunization of the newborn (especially in the case of whooping cough). Key points here include:

Newborn IgG levels correlate with maternal IgG levels (i.e., the active transport mechanism isn't saturated in most cases). This suggests that low IgG for a particular antigen in the mother will result in low IgG for that same antigen in the newborn.

Antibody transport begins as early as week 14, but both the total transport and the rate of transport increases over the length of the pregnancy. This suggests an hypothetical optimum time for an intervention that would temporarily boost the level of any antibody.

Pertussis example
Whooping Cough, or pertussis, the illness caused by Bordetella pertussis, an encapsulated bacteria, is quite dangerous in the newborn. A few years ago recommendations for vaccination against pertussis were updated to immunization in the third trimester, regardless of the mothers prior immunization status. This recommendation is based, in part, on the understanding of transplacental transfer of IgG described in the article I linked in the earlier section, and in part on data on the association between timing of maternal immunization and fetal, all in the context of increased pertussis case rates.
Shortly after these recommendations were updated, additional, more thorough data confirmed this recommendation, though it suggested a slightly narrower window for the ideal time of Tdap administration (from 27-36 weeks to 27-30 weeks).
An aside re: the relationship between passive immunity of the newborn and childhood immunizations
Passive immunity from antibodies transferred to the fetus during pregnancy declines pretty sharply after birth. By 8 months the maternal IgG contribution is effectively gone. This is passive immunity because only the products of B-cells (the antibodies) are transferred. Immunization of the child, on the other hand, provides active immunity, because they develop the capacity to produce their own antibodies. I'd refer you to the allergy and immunology chapter of Bernstein and Shelov's Pediatrics for Medical Students, a surprisingly good general reference.
How should we interpret the linked article in the OP
This article is a comment on a very interesting study in baboons. It doesn't present any new data, but does comment on epidemiological data from a natural Swedish cohort between the discontinuation of whole-cellular pertussis vaccine 1979 and introduction of acellular pertussis in 1996. They reference this data through a misinterpretation of yet another study that analyzed infection rates between 1986 and 2007. The misinterpretation is as follows:

The (1996) drop was pronounced in infants too young to be vaccinated (Fig. 1A), itself a clear indication of herd immunity.

This is incorrect. That figure represents the decrease in infection rates for infants under 1 year of age at the point when vaccine was re-introduced. This included infants who were vaccinated (at 3 and 5 months).
The data before the re-introduction of vaccine, e.g., in 1995, though, does provide a good natural experiment for the epidemiology of pertussis in a non-immunized and immunization remote population. It had been 16 years since the use of whole-cell pertussis and you can clearly see the waning immunity in the population incidence as a whole. If you are really interested in digging into these questions, this cohort is a good place to start. Sweden is very good at collecting important data, so it may be out there.

Hello Dan, this is a good amount of information. However, while I agree that prenatal vaccination offers the best protection, the question I asked is if it is reasonable to conclude that childhood vaccination would result in high enough IgG levels to result in ANY maternally derived immunity.

Thinking about it, based on evidence that even after five years after the vaccination, there are still significant IgG levels and we've concluded that Pertussis antibodies can be transferred, it seems reasonable that, while maybe not enough to provide full protection, childhood vaccination could influence infant susceptibility to Pertussis infections.

@DanielGoldman both remote maternal immunization and maternal infection are considerations. Your question is very interesting. I'm not fully able to answer it, but there are probably data out there that could help. I've edited my question to address the article you linked, but I don't think that's going to be completely satisfactory :)

That's fine. The information that you've provided has been helpful. Maybe the lack of a complete answer is a suggestion that research should be doe on the topic. Shame I don't run a lab. I'd get right on it.

Is it reasonable to suppose that if the vaccine is still effective and
  is causing the body to produce antibodies,

Vaccines do not cause anyone to make antibodies their whole life.  Eventually, the antigen in the vaccine is cleared away, and the immune system stops reacting.  But memory cells have been created, which are ready to quickly re-active the immune system if the antigen is encountered again.
Think about it.  Can you name any childhood diseases that are known to usually skip a generation?  People lived for thousands and thousands of years with no vaccines, and everyone got every disease their mother got as a girl.

Vaccines do cause people to make antibodies their entire life. Long lived B-cells continue to secrete antibody at a low level. There is a not inconsequential circulating level of antibody present long after exposure to some antigens. Certainly the passive immunity from this circulating level of antibody is not the same thing as the robust response from an expanding clone of memory cells, but it is there, and is the basis for, among other therapies, IVIG infusions for primary immunodeficiencies. If you want to edit this answer, I'd be happy to upvote it.


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What is the developmental and health outcome of an XY individual with an SRY addition on their X chromosome?
I know that "XX male" syndrome occurs through various means, including the addition of an SRY gene in one of the X chromosomes. The developmental and health outcomes of such individuals can be problematic. But what about a person with an X chromosome with an SRY addition, along with a Y chromosome?
It seems that it would be similar to XYY syndrome, but without the over-expression of anything but the SRY gene. Would it even be obvious, from a visual point of view or fertility point of view, that a person has the extra SRY gene?
I know that rodents aren't humans, but it does appear that there are species that have naturally acquired multiple copies of the SRY gene.

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Why do mushrooms grow in rings (sometimes called "Fairy Rings")?
Mushrooms disperse their spore in all direction. Then why is there a formation of fairy rings? Why do they grow in one shape and not randomly?

Welcome! I think this is an excellent question! :)

The mushrooms require a lot of nitrogen, more than grass, and they deplete the nitrogen enough that the grass withers, so they grow outwards. https://en.wikipedia.org/wiki/Fairy_ring#Necrotic_or_rapid_growth_zones

When spores germinate, they do not generate mushrooms directly, but haploid mycelium starts to grow. Only when the mycelium from spores with the other mating type is met, cells fuse and generate diploid mycelium, from which mushrooms can grow.

Fairy rings are caused by a radial outgrowth pattern of the diploid mycelium, followed by resource depletion in the oldest part (middle).

This page seems to have good additional information about hyphal growth.

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Plasmid in the nucleus and gene expression
If we insert a plasmid into a human nucleus that contains exact copy of gene and all relevant promoters to produce some human protein,  will the cell create functional protein from that plasmid only when the cell needs it?
I am not talking about inserting DNA to some chromosome. Inserting separate plasmid to the nucleus (transient transfection).

If you transfect cells with plasmid then these plasmids need to go into the nucleus (otherwise they wouldn't be transcribed). Getting the plasmid either happens during cell division (when the nucleus is not present) or by adding a signal sequence to the plasmid which induces the import into the nucleus through nuclear pores. The SV40 sequence is an example of such a sequence.
If you have the right promoter present on the plasmid, you can basically express any protein in the cell (unless it's toxic). The promoter either needs to come from the organism from which the cells are or has to be something universally activated like the CMV promoter (from the cytomegalo virus).
A good example for this is the expression of green fluorescent protein (GFP) which originates from jellyfish and can be expressed in cells to make them glow green (from here):

Untransfected cells or cells transfected with an empty plasmid (without GFP) don't glow.
Targeting specific cells or control the specific activation of genes only in one cell type is possible. For both you need sequence (and cell specific) promoter sequences which either allow the nuclear import (see reference 1-3) or the expression from a cell type specific promoter (see reference 4).
This has been done in the liver of mice in which the ApoE has been knocked-out. The expression of the human ApoE gene with a specific promoter led to the correction of the hypercholesterolemia the mice were suffering from. This needs a lot of knowledge on the promoter as well as on the other transcriptional regulation elements in the specific gene (see reference 4).
References:

Cell-specific nuclear import of plasmid DNA.
Tissue-specific and transcription factor-mediated nuclear entry of
DNA.
Progress and prospects: nuclear import of nonviral vectors.
Sustained liver-specific transgene expression from the albumin
promoter in mice following hydrodynamic plasmid DNA delivery.

Thank you, Chris. I edited the question, it may be not clear, I mean some human protein, that existing in normal human cells. Lets say - the DNA that code for that protein is damaged and now we inserting plasmid with working one. So will the cell create now working protein too, and only when the cell need it? Thank you again.

That's harder, I will edit the answer.

@chris GFP comes from Quail? I thought it came from a jelly fish. And nuclear entry isn't easy, plasmids don't just enter the nucleus. In rapidly dividing cells, they can enter when the nucleus breaks down during mitosis. Getting them to move through the nuclear pore complex is tougher.

@user137 You are right, I used the wrong word. Also about the entering of the nucleus. I edited the posting, thanks for the correction.

@Chris The SV40 sequence is an interesting one. David Dean has done a lot of work on cytoplasmic transcription factors binding to plasmid and pulling it into the nucleus. My boss doesn't believe it, I'm undecided. Quite a few papers about it, but I haven't tried any experiments myself. I suspect the transfection agent used can alter how well it works, because if your PEI or lipofectamine or whatever doesn't dissociate, the transcription factors might not be able to reach their binding sites.

@user137 Interesting thought. It might then also be possible that the transfection reagent helps going through the nuclear membrane as well.

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Non-harmful bacterial invasion of cells
There are bacteria that can enter body cells as parasites.
Could it be that some of these are benign, such that the guest will not kill the host cell it lives in (especially in human)?

I think lactobacillus might be a good answer here

@ChrisStronks, thanks for the edition. Yes I tried to google it, didn't find good answer. And yes, it's really the direction of mitochondria, if there is something like that, at least not harmful to human.

@Barbos, no worries. Interesting thoughts. +1

@rhill45 Lactobacillus don't enter cells IIRC. I think some Wolbachia infections of insects may be possible, not very sure though.

@MarchHo he did say that sorry missed that. Driving and iPhone and SE prob bad idea

@MarchHo, according to wiki and few others, Wolbachia don't lives in human, only insects, and I am asking more about humans. But still, very interesting, thanks.

@Barbos Hmm, I didn't see the "in human" part, so apologies for the irrelevant example.

@MarchHo, it is relevant, helpful and interesting! Thank you. Also I added "in human" a little bit after posting the question - so I need to say sorry.  Just already heard before, that in some spices there are some symbiotic relationships with microbes (inside cells), but it's good to see real example. So again, thank you!

You wish to limit the examples to bacteria?

@canadianer, what else possible? (not talking about viruses of course)

Isn't this how mitochondria came to be a part of our cells?

Why just humans??

@WYSIWYG because I already know that there is such thing in other spices.

@Anne What you are referring to is called endosymbiontic theory.

Plasmodium vivax was historically used to induce fever as a treatment for syphilis (malariotherapy).  Not exactly benign, but it had a therapeutic benefit nonetheless.

Survival of the host cell is in many cases dependent on the egress strategy of the pathogen. There are many examples documented in various species. The only case I found that's somewhat relevant to human cells was were mutant Legionella pneumophila, defective in the lytic pore complex, did not produce necrosis of the host cell. However, the cells did release the bacteria after a while, probably apoptosis due to accumulation.
EDIT: I found an article documenting Cryptococcus neoformans infection.

Cryptococcus neoformans causes disease in people with immune deficiencies such as AIDS. Upon infection, C. neoformans cells are ingested by macrophage immune cells, which provide a niche for survival and replication. After ingestion, macrophages can expel the fungi without causing harm to either cell type, a process named nonlytic exocytosis.

I couldn't find any examples in humans (aside from mitochondria, if those count), but there are examples of varying degrees of intracellular endosymbiosis in insects, plants, and single-celled organisms.
An example is Ca. Carsonella ruddii, an endosymbiontic bacterium of psyllids (a family of plant-feeding insects).  The bacterium lives within specialized host cells called bacteriocytes.

Many insects harbor intracellular bacteria that produce and excrete essential amino acids into the host cytosol. Although, in some cases, ‘dead-end’ bacterial cells with incomplete genomes are replaced by new endosymbionts, this is not the case for the bacteriocyte Carsonella ruddii. The genome of Carsonella is reduced to a mere 160 kb (within the range for organelles), has lost all genes for many essential functions, and no other bacterial endosymbionts are present to offer potential compensation. Thus, Carsonella seems to be at the end of an endosymbiotic continuum in sap-feeding psyllid insects and represents the recent acquisition of a new type of organelle for biosynthesis of amino acids.

Source: The intracellular cyanobacteria of Paulinella chromatophora: endosymbionts or organelles?
Wikipedia's article on endosymbionts has some other interesting examples.

there is a word symbiosis which means interaction between two different organisms living in close physical association, typically to the advantage of both.Often, especially with microbes, one organism lives inside another — the host.
symbiotic microbesWhen both organisms benefit from the relationship, it is called mutualism. When only one organism benefits, but the other one is not harmed, it is called commensalism.
and yes,bacteria can have symbiotic relationship with human.bugs inside
One example is the bacteria that live inside the human digestive system. These microbes break down food and produce vitamins that humans need. In return, the bacteria benefit from the stable environment inside the intestines. Bacteria also colonize human skin. The bacteria obtain nutrients from the surface of the skin, while providing people with protection against more dangerous microbes.
moreover Your body is made up of around ten trillion cells, but you harbour a hundred trillion bacteria. For every gene in your genome, there are 100 bacterial ones. This is your ‘microbiome’ and it has a huge impact on your health, your ability to digest food and more. microbiome


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Production of processed Insulin in bacteria
Genetically-engineered bacteria are used to produce insulin in industry, but as far as I know, the bacteria can produce only proinsulin. Why is that? What happens in the human body in order to make functional insulin, that can't happen in some microorganisms? In the body proteases cut some at amino acids, and cleave the polypeptide while other enzymes create disulfide bonds.  Why is there no bacteria in the industry that can do the same? It's not possible for some reason or in other words — can such a bacterial strain not be engineered?

Have you read the Wikipedia article on Proinsulin? This has an explanation, can you say, which part of it you don't understand?

@Chris, yes. I don't see there answer to my question - why genetic-engineered bacteria could produce only proinsulin and not functional insulin? In the body proteases cut some amino acids, and then enzymes creating disulfide bonds. Why there is no bacteria in industry that can do the same? It's not possible for some reason?

Because insulin is held together by disulfide bonds, which are a pain the ass to properly form in a bacterial expression system. We tried to make bee venom PLA2, which has 5 disulfides, in e coli, and even following a published protocol for recovering the misfolded protein from inclusion bodies, unfolding it, refolding it with glutathione, our yield was terrible. Trying to mutate any of the cysteines ruined the refolding and all enzyme was inactive. Producing proinsulin is just easier at industrial scale.

@user137 Thank you! But why is it so hard to get right disulfide bonds in bacteria? Isn't it work of some enzyme?

@barbos The cytoplasm is a reducing environment, where disulfide bonds are likely to be reduced to free thiols. Disulfides survive better outside the cell. This is true for eukaryotes too, the proinsulin doesn't get converted to insulin until it's in a secretory vesicle, protected from the reducing environment.

@user137 this seems to be good enough for an answer to me

@rumtscho Answer is written. Made a comment initially because I don't really know the details behind industrial insulin production and someone from Eli Lilly or Norvo Nordisk would be better suited to answer it, but I don't think they would.

@user137 sure, details are always great. But a partial answer is still welcome, especially when it contains knowledge novel for the most readers. And I doubt that many of the site visitors have tried protein synthesis in bacteria, so something which might be everyday knowledge to you (that disulfide bonds don't work well) is likely to be valuable new information for many.

Insulin is processed from proinsulin by folding the peptide and forming 3 disulfide bonds before cleaving the peptide in 2 positions, leaving 2 peptide chains held in place by the disulfide bonds. This process is summarized in this image:

This only works because the peptide is packaged in a secretory vesicle during synthesis. This protects the peptide against the reductive environment of the cytoplasm, where glutathione is likely to reduce the disulfide bonds that hold insulin together.
When industrial insulin production moved from extracting the hormone from animal pancreases (pancreai?) to bacterial production of human recombinant insulin, glutathione was likely to cause a problem because simple protein production in bacteria just builds the protein in the cytoplasm, where glutathione is present. It is possible to export the protein into the periplasm during synthesis, similar to how proinsulin is moved into the endoplasmic reticulum during synthesis in human cells, but this can complicate protein purification (larger volumes of media, if protein is inside cells you can spin the cells down and work with a smaller volume).
Note that more recent methods use yeast to make insulin, potentially allowing production of actual insulin instead of proinsulin. And modern purification methods can extract insulin from animal sources that is just as good as recombinant sources.
I know from personal experience that it can be very hard to produce proteins with multiple disulfide bonds in bacteria. We attempted to produce mutants of bee venom PLA2 in E. coli, but the protein was misfolded and wound up in inclusion bodies. We purified the inclusion bodies, unfolded the proteins, then slowly refolded them by dialysing the protein in a glutathione solution. We aimed at making a protein with a free thiol for later chemical modification, but that free thiol just picked up a glutathione we couldn't remove without ruining enzyme activity.
Also please note that I am not fully aware of how insulin is made on the industrial scale and I could be wrong. That's why this was originally a comment.

As far as I know, the extraction from animals plays no role anymore - the process completey relies on production by GMO.

I think some poor countries still use animal insulin, the wikipedia page also says animal derived insulin is used for pets with diabetes. Probably cheaper to give cats and dogs cow insulin than to start full scale recombinant production of cat and dog insulin.

@user137, again thank you very much! You wrote about moving insulin to periplasm. So is it possible to pump hormone completely out of the bacteria? To the medium that it lives in. And we will get a final product - working insulin?

@Barbos Yes it should be possible to secrete it. B subtilis is often used for that purpose. Purification shouldn't be harder, just different.

@canadianer, thank you. I found just that article - http://link.springer.com/article/10.1007/s00253-003-1289-4. There is again production of proinsulin and not the working insulin. Maybe you can point me to the right source?

@Barbos I was only speaking hypothetically. Proinsulin processing is enzymatic, which complicates things.

So what, hypothetically, need to be dune? If I understand right - we can pump proinsulin to the periplasmic space, and it isn't a reducing environment, right? So the next step is to pump to the same area enzymes that will create a disulfide bonds. Am I right? Thank you!


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Telomerase as cancer target
There are a lot of publications, starting from 2000, about using telomerase for targeting cancer cells (it is upregulated in more than 80-90% of tumor cells). Specifically using its promoter (hTERT). In all publications, researches talking about the success of this approach.
So my questions,  why there is no use of this? What is the problem? I read a lot of articles - and didn't find an answer.
http://scholar.google.com/scholar?q=telomerase+suicide-gene+promoter
http://www.spandidos-publications.com/ijo/21/3/661  (one of the newest)

The study you linked to uses cell cultures; there's a big difference between introducing genes in cultured cells and in whole organisms. Also, telomerase is expressed by some non-cancerous cells in adults.

@canadianer, thank you. But in studies that I read, they wrote that the targeting is really good, and has a really little affect on healthy cells - much better then chemotherapy. So the delivery is the main problem...?

Delivery of a transgene would be a problem currently. Delivery of some inhibitory drug could be easier.

Thank you. If you want to write an answer - I will choose it.

With help of canadianer (thank you) I think I found the right article, so just want to share.
This study not talking specifically about telomerase, but about the same approach of using promoters that are upregulated in tumors - exactly what I looked for. Published in 2014.
http://symbiosisonlinepublishing.com/genetic-science/genetic-science03.pdf

However, the therapeutic approach in humans is limited by
  low efficiency of delivering the suicide genes and concerns regarding
  safety. Thus the number of clinical trials in human patients has been
  limited and treatment benefits observed modest. An intensive research
  effort is necessary to overcome these obstacles and turn suicide gene
  therapy into real benefit for our patients.


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Why oxLDL accumulate to form foam cells?
In atherosclerosis, why macrophage store all the cholesterol from oxLDL inside, and turns to foam cell, and not just degrade it and going back to blood? (there are some amount that leave the plaque, but much more stay (J Clin Invest. 2011 May;121(5):2025-36. doi: 10.1172/JCI43802. Epub 2011 Apr 18.))
Reading a lot about atherosclerosis, but no explanation about why macrophages stay there and form a plaque, instead leave or at least break the cholesterol.

If you need more information about oxLDL provide more details about what you need out of your question so answers would be more direct and problem solver and if one answer is not what you are seeking for just comment on the answer

In short, rate of expression of intracellular cholesterol removal proteins like ABCA1 in macrophages and cytokine waterfall molecules in T-lymphocytes beside other anti-inflammatory cytokines has an important role in AS and why oxLDLs accumulate in intima.
When there is high level of oxLDL, rate of removal mechanism must increase to take extra cholesterol out of macrophage and after that to the liver.
And it(increasing the rate of reverse mechanism) could be done by inhibitors or regulators(when natural way could not effort enough excretion). => drugs like TONSHINONE
There is huge amount of information about how regulation of adhesion molecules, costimulatory molecules, antigen presenting molecules and anti-inflammatory or inflammatory molecules could have effect on oxLDL accumulation in macrophages and T-lymphocytes which cause damage induced by hydrogen proxide.
So accumulation of oxLDL molecules in Macrophages has genetic reason and why human being evolved in this way is a question for geneticists but how this happens could be explained by tracing adhesion and signaling molecules.
This is one of the texts about oxLDL accumulation and AS.
Macrophages take in, a large number of oxidized low density lipoproteins (oxLDL) under the intima and then become foam cells, which is one of the early signs of atherosclerotic lesions.
ATP-binding cassette transporter A1 (ABCA1) is an integral membrane protein that transports lipid from the cells to the liver with subsequent lipid excretion by bile at last. This reversal transport inhibits the formation of lipid.
This text shows how regulating cytokine waterfall and pro-inflammatory cytokines could have impact on creation or removal of foam cells.
They experimented that Tanshinone IIA (a molecule extracted from root of Salvia miltiorrhiza) significantly increased expression of ABCA1 in macrophage-derived foam cells, raised the outflow of intracellular cholesterol, and inhibited the formation of oxLDL induced foam cells.

Furthermore, the liver X receptor (LXR) and peroxisome proliferator activated receptor (PPAR) which are members of nuclear receptor superfamily can increase expression of ABCA1 through coupling path. Tan IIA inhibited the formation of foam cells. The mechanism might be that Tan IIA increased expression of PPARa, LXRa, and ABCA1, facilitated reversal cholesterol transport, and ultimately promoted cholesterol efflux.
T-lymphocytes in plaques are mostly CD4+ T-lymphocytes which belong to Th1 subtype. Most of CD4+ T-lymphocytes are active and can induce “cytokine waterfall” as well as secrete proinflammatory cytokines interferon-gamma (IFN-y), IL-12, and other cytokines, eventually leading to inflammation. Experiments showed that Tanshinone significantly inhibited production of Th1-derived IL-12 and IFN-y in a dose-dependent manner. T-lymphocytes play a role in the formation of AS, mainly because of proinflammatory cytokines secreted by T helper type (Th) 1 or Th2. Therefore, Tanshinone can inhibit the formation of AS and stabilize plaques via reducing the secretion of Th1 or Th2. Moreover, T-lymphocytes are activated to express CD40L, and the expression of tissue factor and matrix metalloproteinases (MMPs) is then promoted by interaction between CD40L and CD40.
TanIIA could improve the activity of superoxide dismutase (SOD), decrease the level of malondialdehyde (MDA), and reduce both the expression of CD40 and the activity of MMP-2.
So normally macrophages could excrete cholesterols but problem occurs when your diet contains high level of cholesterol and oxLDL goes high in your blood.
Evidence-Based Complementary and Alternative Medicine
Volume 2014 (2014), Article ID 267976, 6 pages
http://dx.doi.org/10.1155/2014/267976


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Is MHC1 knockout sufficient to prevent transplant rejection?
A few days ago I read about MHC1 knockout pigs for organ transplantation research.
I was just wondering, is it enough to knock out MHC1 in the donor (lets say from same species, pig to pig) for successful transplantation?
I understand that someone without MHC1 is more sensitive to  viruses, cancer, to NK and more. But the study pigs lived fine, so in theory it is possible to have knockout MHC1 and survive.
But is it enough for successful  transplantation from one pig (donor with knockout) to another (fully healthy, normal pig) ?

No, MHCII mismatch can also lead to transplant rejection, though through different mechanisms. CD8+ Tcells (that recognize class I) may become activated and directly kill donor cells*, and some NK subtypes can see the lack of self MHCI and also induce cell death.
MHCII mismatch on antigen presenting cells (APCs) brought along in the tissue may activate CD4+ (helper) Tcells, which do not kill directly. However, they can secrete a lot of pro-inflammatory cytokines which will make a generally hostile environment, and can also activate b-cells to produce antibodies against minor histocompatibility loci.
As to your other question, lacking MHCI entirely would be really rough on an animal in the wild, but under laboratory conditions where pathogens can be managed a bit more, it doesn't matter so much. It's the same reason we can keep mice that have gross immunological deficiencies alive, where humans with the same defects would be screwed.

What about MHCII ? It's not on the organ. Also you talking about " lack of self MHCI and also induce cell death" but that is not the question. Also it's wrong cause pigs live well.

It's rather snide to say "You're wrong they live well" without having ever linked the paper showing this. Without the paper we don't know how far out they were followed, what immunosuppression regimes they may have been given (or what pre-treatment of the organ, etc), or how closely related the pigs were. All of these factors and more can effect the probability of transplant rejection.
Also, the organs almost certainly DO have MHCII  on resident myeloid cells. Invading myeloid cells will also have MHCII and have the potential to initiate an adaptive response against the transplant.

@VGranin - you're right, MHCII isn't on the organ tissue cells, but it's hard to remove all of the APCs from a graft. Modern transplant protocols do work to remove some of them, but it's not 100%. You asked about using MHCI knockouts as donors, I'm saying that an organ that completely lacks MHCI may be targeted by NK cells.

As to MHCI part - you asked about using MHCI ko animals as donors. I'm saying that lack of MHCI on the donor tissue may cause it to be targeted by NK cells. Not sure what you mean by "it's wrong because the pigs live well." Could you clarify (and as @InactionPotential alluded to, link to the study in question)?

http://www.ncbi.nlm.nih.gov/pubmed/25339675

It's an interesting paper, but there are some limitations to your understanding re: they live well. The paper says at the time of publication they were 7 mo old. It does not describe were they are kept, but likely in a highly controlled research facility. For comparison, we keep a number of strains of immunodeficient mice in our facility that wouldn't last a week outside. They are at lower risk of serious infection than your everyday farm/wild pig.

Also, regarding NK cell killing and lack of MHC-I: NK cells undergo a maturation process to a so-called "licensed phenotype" (licensed to kill, ha ha). Immature NK cells are poorly capable/incapable of killing other cells. Maturation into an effective killer involves a process that requires MHC-I expression (based on numerous mouse studies, likely applies here). In animals that do not have MHC-I, the NK cells likely never develop the potential to kill MHC-I negative cells. So they are protected from their own NKs, but licensed foreign NK cells (as in a transplant) could still kill them.

@InactionPotential... Ok, but it doesn't meter how well 100% MHC1 knockout pig lives. Interesting only the transplantation of liver, for ex., with MHC1 knockout to a fully healthy pig. So the NKs of the healthy pig, will attack the liver because it's with no MHC1? But doesn't it will be better than foreign MHC1 that we have now in transplantations?


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Brain regeneration - book recommendation
Searching for an up-to-date book on regenerative brain medicine with a focus on stem cell therapy.
Also interesting in genetic engineering of neuronal stem cells for this purpose.
Alternatively, a comprehensive review article is welcomed too.

I modified the wording - feel free to roll back.

Are you interested in neural regeneration in strictly the CNS, or in the PNS as well?

Neural regeneration is addressed broadly in this chapter from a book published in 2013 (though I would say for a topic such as this, you're probably going to want to read primary literature or recent, less comprehensive reviews in addition to any book chapters published in the few years):
Neural Regeneration by Melissa M. Steward, Akshayalakshmi Sridhar, Jason S. Meyer, published in New Perspectives in Regeneration.
The chapter includes two sections on stem cell therapy and one section on reprogramming of somatic cells to neural lineages.

Regeneration... What a lovely word since 2010
You could start with This text published on 2010 which shows how P21 and it's controller(P53 gene) could regulate regeneration of mammalians' body parts and within them is neurons too.

Regeneration of a hole on ear of MRL mouse or p21 knockout one
This event was accidentally observed ten years before the text get published.
They just couldn't find the ear tagged mice, because there was no hole on their ear anymore.
And you could continue with this text which explains:
p53-Independent Cyclin G Expression in a Group of Mature Neurons and Its Enhanced Expression during Nerve Regeneration
Google Scholar is full of texts, experiments and their results which you need to gadder your information about pathways just by studying and comparing them.
But the most interesting one of them for me was p21 knocked out mice.
As physics helps computation world(Quantum Computers) to do biophysical calculations faster and faster, watching how these proteins and genes play with each other in a 3D simulated human cell like software, will not be a dream anymore.
hope it helps

You've included figures from PNAS, which could get Biology SE in trouble over copyright issues, especially since I do not believe the article in question is Open Access.

PNAS's image is removed and users could have access to it and their related full text through links provided for them while they are open for public if you search on google scholar.


text:
Why shouldn't dogs eat chocolate?
I was wondering why dogs shouldn't eat chocolate. Can't dogs just excrete the indigestible component in their droppings?
It's common knowledge that dogs shouldn't eat chocolate. What I don't know is why chocolate would kill them, from a specifically biological perspective.

Welcome to Bio.SE! I upvoted this question because it generated an interesting answer to me. Generally, for future posts do consider doing a bit more research and explaining what you discovered before posting. This community isn't a "homework service".

Please note that chocolate is toxic also to humans, the difference is that we can tolerate much higher doses. There are also some positive effects of cocoa ingestion so I am not asking anyone to stop eating cocoa-based products. Just adding some context.

Dogs can eat chocolate just like you can eat nightshade: once. Maybe update the question to say "shouldn't"?

@zzzzBov can have (at least) two meaning - being physically capable of and being permitted to do something. E.g. "you cannot drive more then 50 mph in this zone".

@MaciejPiechotka, sure, and "literally" means "figuratively" as well but that doesn't mean one needs to use ambiguous phrasing when better options are available.

@MaciejPiechotka What's your point? My (hypothetical) dog can eat chocolate in both senses of the word. He is physically capable and is allowed to do so (because I'm a (hypothetical) bad owner and I don't care); but he still shouldn't do it.

"Why shouldn't humans eat cyanide? Can't humans just excrete the indigestible component in their droppings?"

@imallett If you don't know why cyanide is poisonous or even that cyanide is poisonous, but do have a basic grasp of the organism's functioning, that's really not such a stupid question. I suppose you're trying to make the point that ingesting a toxin is generally a bad idea, but that doesn't really answer questions centered around why something is a toxin, which this question appears to be. Lots of interested dog owners will be able to tell you that chocolate contains cocoa which contains theobromine which is bad for dogs, but not specifically why theobromine is bad for dogs.

I'm voting to close this question as off-topic because it is trivial. This site is for biological academics, researchers and students, not for people wanting nutritional advice for their pets. Try a vet.

The reason is simple: Chocolate contains cocoa which contains Theobromine. The darker the chocolate is (meaning the more cocoa it contains) the more theobromine it contains. This is a bitter alkaloid which is toxic to dogs (and also cats), but can be tolerated by humans.
The reason for this is the much slower metabolization of theobromine in the animals (there are reports for poisonings of dogs, cats, birds, rabbits and even bear cubs) so that the toxic effect can happen. Depending on the size of the dog, something between 50 and 400g of milk chocolate can be fatal. As mentioned by @anongoodnurse the cocoa content in milk chocolate is the lowest and much higher the darker the chocolate gets.
The poisoning comes from the Theobromine itself, which has different mechanisms of action:
First it is an unselective antagonist of the adenosine receptor, which is a subclass of G-protein coupled receptors on the cell surface which usually bind adenosine as a ligand. This influences cellular signalling.
Then it is a competitive nonselective phosphodiesterase inhibitor, which prevents the breakdown of cyclic AMP in the cell. cAMP is an important second messenger in the cell playing an important role in the mediation of signals from the outside of the cells via receptors to a reaction of a cell to changing conditions. The levels of cAMP are tightly controlled and the half-life of the molecule is generally short. Elevated levels lead to an activation of the protein kinase A , an inhibition TNF-alpha and leukotriene synthesis and reduces inflammation and innate immunity. For references see here.
The LD50 for theobromine is very different among species (table from here), with LD50 as the lethal dose killing 50% of the individuals and TDlo the lowest published toxic dose:

The LD50 also differs between different breeds of dogs, so there are online calculators available to make an estimation, if there is a problem or not. You can find them for example here and here. The selective toxicity makes it even an interesting poison for pest control of coyotes, see reference 4 for some details.
References:

Chocolate - Veterinary Manual
Chocolate intoxication
The Poisonous Chemistry of Chocolate
Evaluation of cocoa- and coffee-derived methylxanthines as toxicants
for the control of pest coyotes.

Very interesting, thanks! I always assumed that dogs couldn't/shouldn't eat chocolate because of their lactose intolerance, similarly to why you might regret giving your cat milk if it hasn't been drinking milk its whole life.

Milk chocolate is rarely a cause of poisoning in dogs because it's the lowest in cocoa powder.

From these articles, I gather that you can, however, give your dog white chocolate because it contains barely any theobromine. Or is that a gross misunderstanding of the article?

@Nzall I think anongoodnurse's answer addresses quite well what you ask about.

@NickT, mostly the Thebromine itself, https://toxnet.nlm.nih.gov/cgi-bin/sis/search/a?dbs+hsdb:@term+@DOCNO+7332 Human have fairly high tollerance to many toxins, some argue it is part of the same resistance to mailliard compounds we evolved after we started cooking our food.

@anongoodnurse It's a bit complicated. Yes, milk chocolate has relatively little cocoa (and thus theobromine), but it is also the sweetest - and dogs like sweet things (unlike most cats, for example, which is why cats aren't in much of a danger of theobromine poisoning despite their lower tolerance and body mass). Dark chocolate would be far more dangerous if the dog actually ate it.

@Luaan - Understood. Chocolate is actually more toxic to cats, but because they don't like sweet things, they are less often poisoned. A dog will usually - usually - not have access to enough milk chocolate to kill it, unless the owner's child is selling candy bars! The point is, milk chocolate is the least harmful of the true chocolates.

I may or may not have been calculating my chocolate LD50 for my bodyweight when I saw that table.  You know, just to be safe!

@CortAmmon Taking the numbers from the other answer, when you weigh around 80kg (just for easier calculation) you would have to eat about 100 ounces (or about 3kg) of cocoa powder or about 1600 ounces (about 45kg) (if my rough calculation is right here) of milk chocolate. Theobromine would most likely one of your smaller problems...

So humans can literally die from eating too much chocolate!? A casual search suggests dark chocolate contains about 10% theobromine, so eating 0.7Kg (1.5 lb) of chocolate will kill the average (70kg) person. That means the name given to the popular death by chocolate recipe maybe wasn't given in jest.

@Bohemian is average person able to consume ~4000 kcal in one go? I wouldn't be surprised if it killed someone even without theobromine.

@Bohemian - the wikipedia article the table comes from suggests that the quantity of theobromine is much lower than 10%: the highest proportion suggested for dark chocolate is 1.4%, or about 0.2% for typical "refined" chocolates.  So the figure is much closer to 7Kg, which seems a lot more reasonable.

@MichaelKjörling actually, not, their answer does not address that point.. It doesn't mention WHITE chocolate, as in the one made using a different end product of the cocoa bean processing. I'm not talking about milk chocolate or any other of the chocolate types they mention.

The toxic ingredient in chocolate is in the mythylxanine class, a substance called theobromine. It is much like theophylline; overdoses of theophylline used to be very common before the advent of inhalers for the treatment of asthma. (Chocolate also has some caffeine in it, which may exacerbate the effects of theobromine.) As @Chris stated, it is only slowly metabolized in dogs.
Generally, toxicity depends on the size, age, the breed of the dog, any medical problems the dog may already have, as well as what type of chocolate was consumed. The larger the dog (given no other predisposing factors), the more theobromine they can handle, and older dogs tend to have more problems than younger ones.
Here is the dose of theobromine that can cause dangerous symptoms, and death:
20 mg/kg (or 20 mg/2.2 pounds, or about 10 mg/pound) may cause agitation, hyperactivity and gastrointestinal signs such as drooling, vomiting, and diarrhea.
At >40 mg/kg, cardiac signs can be seen, and include a racing heart rate, high blood pressure, or even heart arrhythmias.
At >60 mg/kg, neurologic signs can be seen, including tremors, twitching, and even seizures.
Fatalities have been seen at around 200 mg/kg (~ 100 mg/lb), or when complications occur.
Here are the approximate amounts of theobromine per ounce of chocolate:
Cocoa powder: up to 800 mg/oz
Baker’s chocolate (unsweetened): up to 450 mg/oz
Dark chocolate: 150 mg/oz
Milk chocolate: 50 mg/oz
So, theoretically a 40 pound dog (medium-sized dog? My Border Collies each weigh 42 pounds, and are considered medium-sized) will not die if it eats a Giant Hershey's Symphony Bar (a very yummy 6.8 oz / 204g) because that's less than 10 mg/pound. I've never put this to the test, however, and would not recommend it.
Info at https://vcahospitals.com/know-your-pet/chocolate-poisoning-in-dogs

Would you be willing to specify theobromine content in some other, more internationally useful, unit as well? Say, mg/g or mg/kg, or mg/litre, or whatever is convenient.

I have answered the toxicity part in mg/kg, which is an international unit of measurement. There are 28.5 grams per ounce in milk chocolate, slightly more in baker's chocolate and cocoa powder. I have no idea why.

@MichaelKjörling there is a Firefox extension that will convert stuff on the fly. It can be set to show conversions on a menu, or annotate inline, or various other options.  Never fret communicating with your Americqn friends again!  Esp. useful for cooking sites and such where traditional measurements reign supreme.

In this case, I think his list is perfect with the mismatched units!  We have figures for toxicity in mg already, and chocolate candy is sold and sized in oz.  So this allows ready values to be found, looking up and converting in one step.

@JDługosz "chocolate candy is sold and sized in oz" In some countries, perhaps, but not where I'm at. I looked all over the wrapper for a candy bar I had at home, and nowhere does it say oz anything. And of course, I think anongoodnurse's comments that the number of grams per oz differs based on the product being measured is proof enough that such information helps international readers!

I had a chocolate loving dog. She was 12 lbs. The vet said she could have it, provided she wasn't given too much. So his rule of thumb was not to feed her more than I would allow the average 2 year old in 24 hours. Which is two pips ( or two pieces from Hershey's bar). She lived to 16 yo. So yes, chocolate is a hazard to dogs, but like all things done in moderation it should be fine. Check with your vet.

Chocolate contains substances known as methylxanthines (specifically caffeine and theobromine), which dogs are far more sensitive to than people. Different types of chocolate contain varying amounts of methylxanthines. In general, though, the darker and more bitter the chocolate the greater the danger....For instance, 8 ounces (a ½ pound) of milk chocolate may sicken a 50-pound dog, whereas a dog of the same size can be poisoned by as little as 1 ounce of Baker's chocolate!

Chocolate Toxicity Meter for Dogs

Could you support your answer with any credible online reference? Almost all the time answers on Bio Stack Exchange are supported with reference(s) so that the readers can verify the claim put forward by the answerer. And accordingly upvote or downvote. One can also comment on (others') posts just to share and not to answer, but it will require them 50 reputation points which one has to earn by writing Qs and As (which are eventually upvoted as a sign of appreciation {I think}).

Obviously, depending on the dosage you can feed dogs some chocolate without outright killing them. They might have some digestion problems, vomit, or look just fine even though cocoa is toxic for them. But why on Earth would you do that? What are the benefits?

@Tytoalba: To be fair, it could be hard to find sources which show that it is not toxic, even if it were harmless. It would be like trying to find a source which plausibly states that strawberries are not toxic for humans.

@okolnost - Do you like grilled meats, say, a steak? Do you know how many co-carcinogens grilling a steak deposits on the surface of your steak? Sure, you might not get cancer right away, but why risk it at all? What are the benefits? Well, it's exactly the same thing with dogs and chocolate.

Yeah it could be hard. But isn't it done all the time while researching questions which are just not popular and don't show up on simple web results. It is a commonplace for Speceis-ID questions actually. @Michael

OP - I edited your answer to provide support and a source. If the edit is undesirable, please feel free to roll back. :)

@anongoodnurse Similar principle, quite different magnitudes. Also, human deciding for himself about costs and benefits seems to me as different from poisoning a dog on purpose. Are you actually supporting this or is this more of an intellectual exercise (not a bad one, in fact).

@okolnost - It depends on how you look at it. I want my dogs to be happy, so they get treats and all kinds of exercise/interactions. A little milk chocolate on occasion doesn't hurt them any more than running after tennis balls. A little bit of milk chocolate on occasion won't harm the vast majority of dogs. But there are those purists that won't indulge a dog, but will indulge themselves in grilled meats, alcohol, etc.

@anongoodnurse Understood. I would probably try to find a different and healthier treat for my dog (if I had one), as I do with my treats. But I can see your point and I when I think about it I would probably let you take care of my dog (again, if I had one). :-)

@okolnost - Lol at your last comment! Thanks! :D


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Why aren't plants' roots as diverse as leaves?
I have been doing some gardening recently and I suddenly realised that all plants have superficially identical non-woody roots of the same size from gigantic trees to small fruiting plants and vegetables. They are all white, no more than 1 mm across, long and flexible with no distinguishing marks. Only when the root start to become woody do they become different in appearance.
Leaves are very diverse even when they have just started to grow, i.e. before they have reached their full size. I can recognise many different plants from their smallest leaves alone but I wouldn't be able to differentiate plants by looking at their roots. So why aren't roots anywhere near as diverse as leaves?

If you consider rhizomes, corms, bulbs, tubers, nitrogen-fixing nodules, etc. as part of the root system, there's more variety than you give credit for. But an interesting question none-the-less. +1.

@anongoodnurse A potato, which is a tuber, will still put out roots that look like any other plants'. But if you consider that almost every plant can be visually identified by its leaves alone, why not roots?

I was agreeing with you; the roots are very similar. But I wouldn't go so far as to say that every plant can be identified by its leaves alone, if you want me to disagree with you. :)

@anongoodnurse I know you are agreeing with me. I was just saying that, as far as I know, plants with more complex root systems still put out simpler roots that fit my original question. I have personally seen these on rhizomes, bulbs and tubers. I'm not sure what a corm is or what a nitrogen-fixing nodules looks like. I'll have to increase Wikipedia's traffic...

I think the perceived diversity is biased by the relative obscurity of roots and familiarity of leaves as well as the scale at which a human observes these organs with the naked eye. I suspect functional diversity of roots is greater than leaves. One reason for this is that roots compete for and acquire a more diverse set of resources and interact with a larger diversity of organisms than do leaves. In short, if you could observe the metabolome and microbiome I would expect the opposite to be true.

To answer this type of question, we should start by defining 2 concepts: the functions of the root and the external condition that brought to a specific evolution.
Evolution is a necessary process to all organisms, which evolve to optimize a specific function and to better adapt to the external conditions.
There are 3 main and common functions between all land plant's roots: fix plants to the soil; absorb water and minerals; establish specific relationships with other organisms, principally fungi and bacteria. The external conditions of roots (soil) are quite stable, even though, the different "physical" composition of soil, that is the quantity of sand, clay and rocks, change the physical appearance of size and proportion between the main and the lateral roots.Therefore it is plausible to say that it is not necessary to have a great variety in root's structure; since all roots must carry out same functions, all plant's root underwent a similar evolution.
For the leaves it is a bit different. Leaves must also carry out specific and common functions, as: exchange gas (CO2 and O2); contain photoreceptors to "absorb" light for photosynthesis. However, the external conditions of leaves are shortly and continuously changing and highly different (consider the vary regions of the world, as desert and rainforest) with reference to roots. Therefore each plants underwent evolution depending on these types of external conditions, so as to, of course, optimize their functions but at the same time protect themselves as much as possible. Consider, for example, the intensity of sun's radiation, which, if too strong, can destroy cellular structures, the quantity of CO2 in the air, or the high biodiversity of insects, which can carry diseases, ecc...
Interesting book about evolution is: J.C. Harmon, S. Freeman, "Evolutionary analysis", where it is given an idea of the type of evolution, different species, could undergo.
(hope my english is not too bad)


text:
Are there any neurotransmitters that trigger all neurons?
I'm reading into the basics of the nervous system, and am intrigued by neurotransmitters. I understand that certain neurotransmitters can trigger more than one neuron type, and may be used as inhibitors or regulators (antagonists). Conversely, are there any types of agonists that trigger excitation in all neurons, regardless of type? If so, what functions do these play?

There is no such  promiscuous neurotransmitter. There is always some difference between central and peripheral nervous system w.r.t neurotransmitter usage.
Glutamate is one neurotransmitter which acts as an excitatory agonist in most neurons (but still.. not ALL neurons)


text:
How much DNA of mitochondrial origin is incorporated in main cell's DNA?
And especially three points :

in which chromosomes is it located (especially for the human case) ?
how do we know about it ?
does the proportion and composition vary a lot from one eukaryot to another ?

We know about nuclear DNA having a mitochondrial origin mainly in two ways: (1) a sequence in the nucleus is found to closely match a sequence found in mitochondria, or (2) mitochondrial proteins are found to be encoded by the nuclear genome but not by the mitochondrial genome, and those proteins seem likely to have been necessary for sustaining life of the mitochondrial free-living ancestor.
The integration of copies of mitochondrial DNA into the nuclear genome is an ongoing process.  Pieces of DNA can float around and occasionally be spliced into a chromosome.  Most such integrated pieces are entirely nonfunctional (the functional version remaining in the mitochondria) and can pretty much be considered junk despite being replicated when the chromosome is replicated.  Useless gene-like pieces are often called nuclear mitochondrial pseudogenes or NUMTs (see, e.g., pseudogene.net).  NUMTs are of technical importance because they interfere with genome studies: the sequences of the NUMTs can easily be mistaken for true mitochondrial DNA leading to false conclusions about mitochondrial genomes.
A more general term is endosymbiotic gene transfer, which can be used to describe nuclear DNA which was derived from DNA originally residing in mitochondria but now is no longer found there.  Some of this is nonfunctional, but much is functional, coding for proteins which after translation are sent to the mitochondria.  In fact, the overwhelming majority of mitochondrial proteins are coded by the nucleus, many of which probably had a mitochondrial origin.
Eukaryotes do vary in how much nuclear DNA is of mitochondrial origin.  A majority of functional genes are probably ancient and so tend not to vary a lot between organisms.  On the other hand, nonfunctional NUMTs are often relatively recently established and so often vary widely between organisms.
Particular nuclear chromosomes are not associated with mitochondrial origins; the integration of free floating DNA pieces is mostly a fairly random process.
For more detail, you might want to see Timmis et al's review, "Endosymbiotic Gene Transfer".

Interesting question. I researched this a bit now and the phenomenon is termed "numt" for  "nuclear mitochondrial DNA". This term descrives the transfer of cytoplasmic mitochondrial DNA sequences into the separate nuclear genome of a eukaryotic organism. It seems that most of these sequences are inactive. This list at pseudogene.net has a large number of sequences in a lot of different organisms, for humans insertion between 0,2 and 14,2 kb are listed. Most of these sequences are detected in the deep sequencing projects underway these days and by comparative sequence analysis. An example is this paper, where they found mtDNA on chromosome 1 of homo sapiens, which was still part of the mitochondria in homo neanderthalensis.
For more information you can go to the list of papers I list below, this should be a good starting point:

http://www.ncbi.nlm.nih.gov/pubmed/11369110
http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020273
http://www.ncbi.nlm.nih.gov/pubmed/15014143
http://www.ncbi.nlm.nih.gov/pubmed/18721785

The papers should all be accessible, if not, let me know.

Thanks a lot for these pointers. I had no accessibility troubles.


text:
Does dairy negatively affect the voice for singing?
Singers and actors are warned not to consume dairy, primarily milk, before a performance. From personal experience, I know that this is a very valid warning. It is commonly assumed that this is due to milk causing either more or thicker phlegm.
Has the mechanism behind this been studied and verified?

As it turns out, dairy products do not produce phlegm in the majority of people (the exception is the tiny group of people who are allergic to casein, the protein in some types of milk). Instead, the high fat content in dairy products thickens the mucous that is already present in a person’s airway, making it seem like there is more phlegm to deal with. Of course, having thicker phlegm can be just as problematic as having more phlegm. Luckily, this thickening sensation can be diminished simply by eating dairy products that have a lower fat content.

(https://www.operapulse.com/refine-your-craft/guide-to-opera-training/the-rules-of-singing-mythbusters-edition/)
Regarding the above quote, there is no medical evidence that states milk consumption causes phlegm production even for casein allergic individuals.
The study cited at the end shows that there is a correlation between milk consumption and thicker phlegm. However correlation is not causation. The authors explain that the allergy-like effects of milk are produced only when tissues are actively inflamed and exorphins (peptides produced when milk is broken down) stimulate mucus production from gut glands. The thicker phlegm effect is shown in only a segment of the studied population. Unless you're one of the few with inflamed tissues, you should be ok with drinking milk before a performance.
(http://www.researchgate.net/profile/Jim_Bartley/publication/40026300_Does_milk_increase_mucus_production/links/0fcfd5138d5212ef80000000.pdf)


text:
Why hand washing causes skin to dry?
This seems counter intuitive: dry hands mean low humidity, so why when humidity is increased, by washing hands, they become even more dry, eventually?
In particular, why such hand dryness is usually observed in a dry climate?

What did you find when you googled the question? What is it about the answer (which you should summarize in your post) that confuses you? Do you understand the role of oils in skin health and hydration?

I washed my clothes in India using clothes powder. my hands were peeling after 2-3 months. i used moisturising lotion. they were fixed.

@Christiaan. I disagree and think the question is relevant here.I  think a biological  answer about membranes, body oils, skin characteristics etc.  would be a  useful response

I will assume the hand washing you speak of involves soap.
The human skin (which also covers hands) is a complex organ with an underlying system of glands.
One of these is the exocrine sebaceous gland which is responsible for the production of a substance known as sebum. This substance is primarily composed of triglycerides, wax esters and other fatty acids. Sebum makes human skin waterproof as it's constituents are not water soluble (oily).

The action of soap on oily substances typically involves increasing their solubility in water so that they can be easily rinsed upon being dissolved. So when one washes their hands with soap they remove the oil layer secreted by the glands under their skin.
Removing the oil layer makes the skin susceptible to drying up as oil reduces the rate of evaporation of water on the skin to maintain a moisture balance. This is the reason why most brands of lotion have a moisturizing effect (or marketing statement).

Good that you mentioned lack of oil,  but to really answer the OP's  question, I think you need to extend your answer to discuss WHY lack of sebaceous oils leads to skin drying out

The temperature of the water can also affect skin dryness. Hotter water increases solubility of oils, and in turn contributes to dryness.


text:
Which organs can be donated after clinical death?
The information I found about organ donation does not address clinical death. For example:

The process of donation takes place only after physicians declare a
  person brain dead [...] cessation of brain function [...] is
  irreversible. However, the vital organs (heart, lungs, liver,
  pancreas, intestine and kidneys) can be kept viable for a few days, if supported by artificial mechanical means (i.e. a ventilator).
  (source)

However:

Blood circulation can be stopped in the entire body below the heart
  for at least 30 minutes, with injury to the spinal cord being a
  limiting factor. Detached limbs may be successfully reattached after 6
  hours of no blood circulation at warm temperatures. Bone, tendon, and
  skin can survive as long as 8 to 12 hours. (source)

Assuming the organs were not damaged by the cause of death itself, are there any organs that can be transplanted after clinical death?

It depends on why the patient died, and how long they were dead before the opportunity arises to harvest the organs. If you want to modify the question to narrow the scope, it is answerable. Death from a head injury on the highway or in the home - without any other significant injuries - provides the maximum potential for organ donation, with time being a critical component.

@anongoodnurse I'm actually interested in an answer covering the parameters you mentioned, assuming the organs were not damaged by the cause of death itself.

As the question stands, I think it might be too broad.


text:
Authority on Microbiological Definitions
Is there an authority on definitions for molecular microbiology concepts, like an IUPAC book for chemical definitions?
The particular definition I am debating is chromatin. Some say it encompasses DNA, RNA and protein, whilst others say that it contains DNA and only during the mitotic interphase can it transcribe to produce RNA and protein.

What you are asking about is molecular biology, not microbiology.

So in a very broad definition, yes, chromatin encompasses DNA, RNA and protein. The primary function of chromatin is to provide a method to effectively package all the DNA in a cell. This packaging is dynamic, and it's constantly regulated in a cell type-specific manner. We can see the broad interactions of protein and DNA in this cartoon representation from wikipedia:

Combined with histone-modifying complexes, and chromatin remodeling complexes, we obtain the aforementioned mode of regulation. Histone protein can be modified (acetylation, methylation, glycosylation, phosphorylation, etc.), they can also be moved, substituted in their subunits for specific pruposes (think H3.3 or H2A.X). We can also end up with varying regions of dsDNA, 10nm or 30nm fiber.
The RNA comes in as a structural and functional components of the chromatin, participating in many controls as noncoding RNAs.
I believe most of the method in defining chromatin comes in very much as the amassed literature on the subject to-date. You'd even consider it "common knowledge" entering the field (i.e. a quick google search can return you the right definition).

"only during the mitotic interphase can it transcribe to produce RNA and protein."

Transcription halts entering M phase or mitosis, transcription may occur during G1, S, G2, or "interphase."


text:
How is the type of genetic manipulation determined in CRISPR-Cas9?
I've been reading up a bit on the CRISPR-Cas9 system for gene manipulation. From what I read, it introduces double-strand breaks at specific points determined by the choice of sgRNA. But how do you get from double-strand breaks to editing genes in incredibly specific ways? As far as I read you can fix point mutations, insert whole genes or disable genes with CRISPR-Cas9. How do you specify which kind of repair mechanism is invoked?

But how do you get from double-strand breaks to editing genes in
  incredibly specific ways?

You should read the wikipedia page on CRISPR-Cas system. The target specificity is provided by the tracrRNA.

How do you specify which kind of repair mechanism is invoked?

The repair can be done either by Non Homologous End Joining (NHEJ) or Homologous Recombination (HR). If you provide excessive DNA template by transfection then it is likely to increase the chance of HR and this can be used for trangenesis.
When no template is provided then DNA can be repaired by HR with sister chromatin or NHEJ. Depending on the stage of the cell cycle one of these mechanisms is preferred over the other. HR is shown to be the most active in S-phase and starts declining in G2-phase.
So when you want to make transgenics or specific edits then co-transfect CRISPR-Cas with transgene DNA (with flanks homologous to target DNA sequence) or a homologous DNA with point mutation(s), preferably in cells at S-phase. It is possible to synchronize the cells using nocodazole or other molecules that arrest cell cycle.

I assume "sgRNA", "tracrRNA", and "guide RNA" are all the same thing? And I can imagine getting efficient editing would be a problem, since you have to get at least 2 components into the cells, the Cas gene and guide RNA could be produced by 1 plasmid, but the template DNA to direct the repair would be separate, and you'd probably want several copies per cell to improve the odds of a successful repair.

@user137 yes pretty much the same thing.. It is not that difficult; transgenesis rates are much better than in plain HR. All these kind of techniques are followed by screening and selection.

My thinking comes from in vivo work, where you'd either have to package all the parts into 1 particle, or get get multiple molecules into the same cell. Though I've seen a paper where the corrected cells are accidentally selected for in vivo.

@user137 You transfect the ESC and then implant them in the ICM right? There is still scope for artificial selection.

@user137 If you want to learn more about the Cas9 system, I wrote a detailed explanation of the original publication by M. Jinek on my blog: http://www.qmviews.blogspot.ch/2015/09/literature-004-jinek-et-al-elife-2013.html
Would be glad to hear if it helped.


text:
Cell line immortalisation biosafety
Cell lines are often immortalised by artificial expression of proteins which specifically cause the knockout of important cancer suppression genes or the activation of proto-oncogenes.

Cellular immortality happens upon impairment of cell-cycle checkpoint
  pathways (p53/p16/pRb), reactivation or up-regulation of telomerase
  enzyme, or upregulation of some oncogenes or oncoproteins leading to a
  higher rate of cell division.

However, this would appear to fall under the purview of Biosafety Level 3 since the protein expression vectors are capable of causing cancer in the researcher when they infect the researcher. Cancer can certainly be considered a serious or lethal disease, and aerosolisation of the vectors, while rare, is not impossible (for example during a spill).
According to the biosafety definitions of Columbia University,

Agents: serious or lethal diseases transmissible via aerosols, e.g.,
  M. tuberculosis, SARS.  Recombinant DNA activities using genetic
  material from BSL-3 organisms or such organisms as host cells.

Why then, does the CDC classify cell line work, including work with viral production cells, as a BSL-2 activity requiring only BSL-2 containment levels, when the act of immortalising the cells would involve working with viral agents that can cause cancer?

Cells immortalized with  viral agents such as SV-40, EBV adenovirus or
  HPV, as  well as cells carrying viral genomic material also present
  potential hazards to  laboratory workers. Tumorigenic human cells also
  are potential hazards as a  result of self-inoculation.[...]
Human and other primate cells should be handled using  BSL-2
  practices and containment. All work should be performed in a BSC,  and
  all  material decontaminated by autoclaving or  disinfection before
  discarding. BSL-2  recommendations for personnel protective equipment
  such as  laboratory  coats, gloves  and eye protection should be
  rigorously followed.

Are the transformation cells (eg the 293T viral cells containing the transformant viruses) considered Level 3 biosafety risk, and if not, why not?

Work with vectors expressing oncogenes or knocking down tumor suppressor genes (particularly if vectors are based on lentivirus) is often done under "BSL-2+" conditions. This essentially means that personal protective measures required for BSL-3 are used, but the specialized ventilation systems and so forth of BSL-3 physical laboratory space are not required. That would seem to address the investigator-safety issues raised in this question. See the complete Columbia policy as an example of the details and the risk-assessment process.
As noted already, once cultured cells have been infected with these vectors the risks to personnel are substantially diminished if not eliminated, so less stringent protections are then required.

Maybe, cells should be contained under BSL-2 and agents (viral particles etc) under stricter BSL-3? That is, after immortalization you can keep cells under lower security.

Biosafety Level 2 builds upon BSL-1. BSL-2 is suitable for work involving agents that pose moderate hazards to personnel and the environment.
Biosafety Level 3 is applicable to clinical, diagnostic, teaching, research, or production facilities where work is performed with indigenous or exotic agents
  that may cause serious or potentially lethal disease through the inhalation route of exposure
CDC Biosafety in Microbiological and Biomedical Laboratories, Laboratory Biosafety Level Criteria

As of HEK 293 cells, check this out:

Biosafety level 2 practices and containment facilities for all activities involving HEK-293 cell lines.
HEK 293 Cell Line Risk Assessment - IBC

+1 for the possible interpretation of the definitions. However, do you have any examples of such viral transformants being actually kept in Biosafety Level 3 environments by an academic research lab?

my guess is that any research group will try to keep as low safety profile as possible (maybe, except NIH labs) to save money, or time, or stress. Low enough for NIH grants and publishing approval.

@aaaaaa : such work is regulated by institutional biosafety committees, which are in the best position to gauge the relative risks of particular procedures and the level of protection required. That required regulation is independent of funding source. Individual labs that do not get permission for the work or do not follow procedures can be shut down. There is more stress in working with such vectors without protection than is generated in the process of applying for permission or for carrying out the protective procedures.


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What is this spider, and what is this object that emerged from its rear end?
I was observing this spider (caught in a Petri dish) under my microscope, when I noticed that a small, round object emerged from its rear end. It appeared to be black and white in appearance. I am guessing that it is an egg, but I am not sure whether it is one or not. The diameter of the object is approximately 300µm.

The spider seems to resemble Hasarius adansoni from photos:

The scale bars in the image are 5mm in length. The spider and the object are imaged together in the frame for scale.

Can anyone conclusively identify the spider and the object? The photo was taken in Singapore in November (although the seasons don't really matter in Singapore).

Excellent images. I wish I could say anything conclusively other than, 'wait and see.' Maybe you'll get lucky and the answer will present itself conclusively.

I do not know what species of spider this is, but I think the white ball is actually spider poop.  Unfortunately a quick search did not return many references, but here is a picture for comparison.

Spiders produce uric acid, which is a near-solid and excreted out white.  This is done to minimize water loss. These malpighian tubules drain into an pouch attached to the digestive tract, or stercoral pocket.  The uric acid waste from the is combined and eliminated together with solid waste from the digestive tract.  In your spiders case, this would be undigested insect parts (black) mixed with the uric acid (white).

Actually, I forgot to update this question, but after 3 days the unknown object was found to be liquefied into a spread around 3mm in diameter. This supports the hypothesis that the ball is spider waste.


text:
Solubility of DNA in water
This would seem to be an easy to answer question, but I was unable to find an answer (in g/L) for generic double-stranded DNA or plasmid neither on Google nor on BioNumbers. I would expect the solubility to vary slightly based on the sequence or length, but it probably wouldn't affect it very much due to the relative hydrophobicity of the bases.
I know I have made solutions of plasmid maxipreps of at least 1µg/µl, and the Nanodrop DNA quantifier datasheet has a maximum DNA detectable limit of 15µg/µl, but can anyone find a source for the maximum solubility level of generic DNA? For example, the solubility of a specific plasmid of approx. 5kb in length would be a good ballpark answer for this question.

I think that we need to dig around for maximum molar concentrations of DNA for this question.  I know that you can't spec a sample above 100g/L (ug/uL), and that to test high concentration giga preps I've had to do serial dilution of the sample for accurate reads.  I've consistently made giga preps that were concentrated to 10+g/L but those were in buffers, not water.

@AtlLED do you have a protocol for the giga prep kit if you used one?

https://www.qiagen.com/us/resources/resourcedetail?id=399337ce-2fb3-495a-b2f3-e35a17f61cab&lang=en  and also http://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NuBoEF.pdf  At high concentrations, the prep becomes much more like a syrup/has an increased viscosity.

This is just a back-of-the-envelop thought experiment: A human cell has  approximately 6 billion bases, meaning that there are app. the same number of - charged phosphate bonds in the backbones of the helices. If you were to say arbitrarily, for argument sake, that you needed 10 water molecules to solvate the negative charge of the phosphate bond, then you would only need 1.8 femtoliters of water to solvate the DNA from a single human cell. The human genome weighs about 3.6 picograms, so if this accurately represented the solubility of dsDNA then it is equivalent to 2g/mL.

This makes sense, because when you think about how absolutely small a cell is and how much water its even smaller nucleus contains, even in this minuscule amount of water, DNA and associated proteins are still solvated, then you get an idea for just how much DNA can be solvated in water. We can easily see small fractions of a drop of 1 µL of water with our naked eye (as you mentioned a nanodrop machine), but we need a decently powerful microscope to see the nucleus of a human cell, and yet all of that DNA is still solvated. It will take a huge number of 5kb plasmids to saturate water.

@AMR Arguably, most of that DNA is bound to chromatins instead of being solvated in water.

Chromatin is linear dsDNA and its associated proteins. Why would that make it any more soluble than naked DNA? Hydrophilic/Hydrophobic interactions will sequester the hydrophobic regions of the molecules from the water molecule. The cell is an aqueous environment, by definition all of the hydrophilic portions of the molecules are solvated in water.

@MarchHo I think that the likely reason that you cannot find an answer to your question is that DNA is a variable molecule. A plasmid will behave differently to linear DNA. A large plasmid will behave differently than a small one. Whether you are dealing with chromatin or whether you have digested the proteins and RNA and are left with naked dsDNA will make a difference. Oligonucleotides are single stranded and will had different solubility. Even small fragments of dsDNA will likely have different solubility than an entire chromosome.

@AMR I asked specifically for the solubility of a 5kb plasmid.

What is your concern related to the solubility of a 5kb plasmid?

The point of my last question was that unless there is a specific reason for needing the solubility of a 5kb plasmid, then the answer of it is soluble in water, is sufficient for almost any application that you will use that plasmid in. It is sort of like with NaCl. Its solubility is 359g/L, for most every application, soluble in water is all that is needed to be known. An E.coli cell is 10^-15L, but they still have enough volume with everything else taking up that volume to replicate tens to hundreds of 5kb plasmids. The point is the DNA, being a charged molecule is highly soluble.

DNA is a bit more complicated than some molecules due to it's length and composition variability.
In water.
According to Integrated DNA technologies:

DNA oligos can be resuspended to a near maximum concentration of 10mMolar; to achieve such a high concentration will require a lot of vortexing and it may take up to a day for the oligo to go into the solution.

DNA is a polar molecule and as far as I have experienced dissolves in water very readily. However there doesn't seem to be an exact quantifiable number for DNA solubility in water. If I correctly understand it, this paper claims that a 200,000Da-8,000,000Da oligonucleotide is soluble in water, however they never quantify this. An answer on a related question pointed out that high molecular weight DNA oligomers, like genomic DNA, is enhanced in solutions with dilute monovalent cations.
Cleaver & Boyer (1971) have some data on this, however it is somewhat outdated, and they were only using this solubility to demonstrate dialysis in water was capable of desalting the oligonucleotides.

Oligonucleotides larger than 5 bases in length remained within the dialysis
bag (Fig. 2) and losses of 10% and 40% occurred for tetra- and trinucleotides,
respectively.

There is a lot of work done on optimal temperature and pH for solubility of DNA so perhaps a more specific query might throw up some results.
In ethanol.
Ethanol precipitates at least 10–20 % of all classes of oligonucleotides. Oligonucleotides longer than 15–16 nucleotides are precipitated to about 65%.

Can you explain why organic solvents are likely to be needed? It seems that DNA is less soluble in isopropanol and ethanol (which are used in DNA extraction) than water, so making the solvent less polar might appear to have the opposite effect.

Hmm... Not sure. It could be to do with the fact that recovering DNA from water could be difficult. I have opened another question for that: http://biology.stackexchange.com/questions/35601/why-do-dna-solutions-contain-organic-solvents

@243 pointed me to a site with the answer you were looking for. I've edited it in.

The link you provide to genscript is for peptide sequences which can have  different solubility properties depending on amino acid composition. Peptides sequences can potentially be far more hydrophobic than DNA.

@InactionPotential You are absolutely correct - this was the wrong server entirely. It is removed and I will re-add it when I find the DNA solubility server.

@MarchHo Other organic compounds are often present because they are used in precipitating the DNA from other molecules in a solution, as a buffer, or for washing detergent from the solution. I think little data is available on DNA solubility because it dissolves very readily, and only the solubility of additional compounds are "important" as they, as you infer, decrease the solubility.

There is a bit of a problem using the IDT number of 10mMolar to think about the solubility of DNA. First off Oligos come single-stranded, so that is a different molecule from a plasmid. Secondly, there is a large amount of salt in the dehydrated microtube they send you. So your resuspension ends up being a buffered solution and not just pure water. Both of these will effect the saturation point.


text:
When did mouth pipetting stop becoming a way to handle liquids in a lab?
Almost all modern lab protocols have an addendum prohibiting pipetting by mouth, instead mandating that a Gilson pipette, a rubber pipette ball, or a serological Pipet-Aid be used.
However, it was clear that some time in the previous century, prior to the invention of the aforementioned tools, many labs did in fact use mouth pipetting to transfer liquids, some of which were highly hazardous. An example can be seen here (from this IO9 article, which dates the photo to the year 1943):

When did pipetting by mouth stop being recommended in labs?

I have done that! And that was 1990s

I stopped mouth pipetting in the '60s.

Although the use of pipettes in the early chemistry laboratories undoubtedly led to accidental aspiration of undesirable toxic and poisonous substances, the first recorded laboratory infection due to mouth pipetting occurred in 1893, the case of a physician who accidentally sucked a culture of typhoid bacilli into his mouth...
It Was Dangerous, Hence was removed... Even though, it is removed, we often see the use of this in some countries..

Late to the party, but apparently "Mouth pipetting" is a thing for single-cell sequencing: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5554467/
In the micromanipulation, single cells are usually aspirated into a glass capillary (typically 30 μm in diameter9) by applying a gentle negative pressure which can be provided by a manual/automated micropipettor (called micro-pipetting) or even a researcher’s mouth (called mouth-pipetting). It relies much on personal skills because the key process of single-cell aspiration should be carefully completed under microscopic field.

Mouth pipetting, while almost unheard of in modern laboratories in developed countries, is still very much a current protocol in many parts of the world.
For example, this paper analyses the proportion of clinical labs in Pakistan and found evidence of poor biosafety practices (emphasis mine):

Results: A total of 1,647 (92.4%) males and 135 (7.6%) females
  participated in the study, with over half (59.7%) having more than
  five years of work experience. Results showed that 28.4% of the
  laboratory technicians from Punjab, 35.7% from Sindh, 32% from
  Balochistan and 38.4% from Khyber Pakhtoon Khawa (KPK) did not use any
  personal protective equipment. Almost 46% of the respondents (34.2%
  from Punjab, 61.9% from Sindh, 25.2% from Balochistan and 85% from
  KPK) said they reused syringes either occasionally or regularly.
  Furthermore, 30.7% of the respondents said they discard used syringes
  directly into municipal dustbins. The majority (66.7%) claimed there
  are no separate bins for sharps, so they throw these in municipal
  dustbins. Mouth pipetting was reported by 28.3% technicians.

Additionally, a Google Scholar search for papers published in the past 5 years involving mouth pipetting turned up 670 hits out of 284000 total hits, a proportion of 0.23%. This is further evidence that this dangerous technique has not been fully eradicated from labs.


text:
Is [O2] different in a 5 mL liquid media?
Is there a drastic difference between top and bottom of the tube, with no stirring?

I believe there is. As you can see in the image below, strictly anaerobic bacteria can survive in liquid medium in test tubes, herefore they will migrate to the bodem of the medium to avoid Oxygen. Also you can see that microaerophiles, which require an oxygen percentage of approximately 2-10% (versus approximately 20% in normal air), will be able to survive below the surface of the medium.
So yes, there is a drastic change, seeing as obligate aerobic bacteria can surive at the surface of the medium, and obligate anaerobic bacteria can survive at the bodem.

To this I have one caveat to add. Namely that these tests are normally done in standard test tubes (approximately 9, 22 or 28mL depending on size), which would most likely contain more medium. So I am not absolutely sure, nor have I found any specific information on oxygen gradients in 5mL tubes. You could test this by performing the experiment in described in the figure in your tubes with 5mL medium and see what happens. Then you will definitely know.
Hope this helps!


text:
Genetically improved *Leucaena leucocephala* seeds
I am looking for fast growing Leucaena leucocephala seeds. Now, I am cultivating Leucaena leucocephala which can grow 6 meters in a year. But I am looking for a genetically improved seed which can grow up to 10 Meters in a year.

I am not saying it is off-topic. But can you repost this on: http://gardening.stackexchange.com/


text:
How can I keep HEK cells alive while expressing NMDA receptors?
I am trying to express functional NMDA receptors in HEK293 line cells for single channel recording experiments.
The HEK cells are maintained in the standard way (Thomas & Smart 2005) and transfected with NR1 and NR2x subunit cDNAs and also GFP, using either lipofectamine or calcium phosphate precipitation. GFP expression suggests successful transfection, but cells exhibiting green fluorescence are uniformly swollen and dead and it's more or less impossible to obtain a successful patch. Untransfected cells seem to remain perfectly viable.
On the basis that the toxicity might be a consequence of Ca2+ influx through open NMDARs, I've tried including a cocktail of blockers in the growth medium, including AP5, kynurenic acid and Mg2+, but the transfected cells continue to die.
Can anyone suggest anything else I should be doing to keep the cells alive? Or am I just on a hiding to nothing? Other researchers seem to have managed this (eg Medina et al 1995, Vicini et al 1998) and do not seem to be doing anything substantially different, so I'm a bit at a loss.

Try perhaps lowering the level of expression of your construct. HEK cells are notorious for expressing large quantities of your transfected insert. Do you have it under a CMV promoter? Try a milder one, such as an ubiquitin promoter or similar or, even better, a doxycyclin-inducible system (e.g., clontech's tet off) so you can manage the expression levels through the dox concentration/time of induction.

It is indeed using a CMV promoter, so that would be something to look at. One suggestion I've seen is to co-transfect with a high ratio of an "empty" pcDNA plasmid as a competitor to reduce expression. That sounds a bit Heath Robinson, but it would be an easier test than splicing into a new vector...

Sure, you can give that a try. I would expect that to lower the transfection efficiency rather than the level of expression per cell, but it's easy enough to try it right away. Good luck! BTW: I had to google "Heath Robinson": nice one!


text:
Are cerebellar basket and stellate neurons actually different cell types?
The title more or less says it all, but to contextualise a bit:
Cerebellar molecular layer interneurons have been classified, probably since Cajal, into the basket cells, which synapse onto the soma and axon initial segment of Purkinje cells, and stellate cells, which synapse onto the dendrites. The types are distinguishable in tissue slices by positional and morphological differences, but it is at least arguable that the variation is continuous and the boundary somewhat arbitrary. Both types take basically the same inputs and deliver the same outputs, albeit in marginally different cellular locations.
As far as I'm aware -- and I would be incredibly happy to be told otherwise -- there is no known difference in protein expression that can be used to distinguish between these interneuron types, for instance in dissociated culture.
So, is there in fact some identifying marker? And if not, is it still useful to classify them as separate types?

You are completely right with your guess that is was Ramón y Cajal who first described these cells in his famous monograph "Histologie de systeme nerveux de l'homme et des vertebres." published in Paris Maloine in 1911.
Since the only method he had at his disposal was Golgi's staining which is a pure anatomical staining (there is no living cell left in the tissue after the treatment with  potassium dichromate and silver nitrate) with silver chromate filling completely single cells at random.
Using only this staining and his eyes Cajal did classify the neurons according to their size, location in the brain and their shape. This is why we have such names like basket, stellate, moss, pyramide etc. in neuron description. This classification is purely anatomical and does not reflect any intrinsic properties of these cell, as for example being motor neurons or interneurons.
When I was studying histology at my medical school I was always told that there is new "mollecular biology"-based classification of neurons is about to come and to replace the existing classification by Cajal, many cell types were supposed to be merged there, among others also different anatatomical groups of interneurons. (This was one of the major research topic of the research facility where I studied neurohistology).
As far as I can see, this classification is still not here...


text:
Identifying the origin of replication of an unannotated *E. coli* plasmid
I have attempted a few searches for a list of origins of replication for plasmids in E. coli, but I was only able to find a list of origins, but not their individual sequences. The available plasmid maps are often extremely vague on where exactly do the origins start or end, and in any case it would be extremely tedious to collate a list of origins in order to determine the exact origin of replication of a plasmid.
Is there a database of origins of replication available anywhere that would allow one to perform BLAST searches on the plasmid sequence in order to obtain the compatability group of the plasmid, in cases where the plasmid's origin of replication is not specified?

What do you mean by "unknown"? Is that naturally generated outside labs? Adgene shows positions of functional sequences and actual sequences.

Plasmapper Is quite good a recognizing common origins of replication. You will have to look up compatibility yourself. It is what I usually use when confronted with unnannotated plasmids.

There must be some such information, because at the lab we are using the SnapGene software, and it is able to recognize the Ori sequences (and all other sequences). Since it is proprietary software, I doubt they would disclose their list, but others might have the same information.
If you really need to find your origin for your plasmid, you could try the demo. Good luck!

Go this URL
And click pBR322 origin in the map. Then, the corresponding sequence will be highlighted.
2248-2867: pBR322_origin
2201    cccttaacgt  gagttttcgt  tccactgagc  gtcagacccc  gtagaaaaga  2250
2251    tcaaaggatc    ttcttgagat  cctttttttc  tgcgcgtaat  ctgctgcttg    2300
2301    caaacaaaaa    aaccaccgct  accagcggtg  gtttgtttgc  cggatcaaga    2350
2351    gctaccaact    ctttttccga  aggtaactgg  cttcagcaga  gcgcagatac    2400
2401    caaatactgt    ccttctagtg  tagccgtagt  taggccacca  cttcaagaac    2450
2451    tctgtagcac    cgcctacata  cctcgctctg  ctaatcctgt  taccagtggc    2500
2501    tgctgccagt    ggcgataagt  cgtgtcttac  cgggttggac  tcaagacgat    2550
2551    agttaccgga    taaggcgcag  cggtcgggct  gaacgggggg  ttcgtgcaca    2600
2601    cagcccagct    tggagcgaac  gacctacacc  gaactgagat  acctacagcg    2650
2651    tgagctatga    gaaagcgcca  cgcttcccga  agggagaaag  gcggacaggt    2700
2701    atccggtaag    cggcagggtc  ggaacaggag  agcgcacgag  ggagcttcca    2750
2751    gggggaaacg    cctggtatct  ttatagtcct  gtcgggtttc  gccacctctg    2800
2801    acttgagcgt    cgatttttgt  gatgctcgtc  aggggggcgg  agcctatgga  2850
2851    aaaacgccag    caac
You can find various ori sequences from this site.

I already know how to do this. I specifically stated that "and in any case it would be extremely tedious to collate a list of origins in order to determine the exact origin of replication of a plasmid. " and therefore asked if there was a database which solves this problem.

It's just four or five in total, isn't it? pMB, modifiedpMB, R6K, p15A, pSC101, modified colE1. If you get a plasmid somebody constructed for recombinant DNA experiments, probably the ori is one of these. If you get plasmids from natural resources, you might not find complete match by alignment, but you could find a similar sequence among them. Or are there more types of ori out there?

I mean once you get those 6 types of sequence, you can blast by yourself. You do not need to search plasmid from the URL I show. Did I miss something?

Try this. I would suggest OriDB also, because it's precisely what you asked for. Getting the sequences out for easy blasting might be a chore, depending.

OriDB is not what I asked for, since they don't have E. coli in their organism database. http://pombe.oridb.org/organisms.php

That's why I didn't list it first. It appears to be the only database of Ori sequences, so it's a pity it is so small.