This is an excellent, but very broad question, Katia! Other than saying things can go up (or down) I don’t think that a lot more can be said about precise neurotransmitter levels, at least in humans. In terms of actual amounts we are sometimes talking about brain tissue levels elevating to 50-100 nanomolar or higher (perhaps from a nanomolar baseline in experimental animals), but this really depends on the technique being used to do the measurements, and the actual neurotransmitter levels at neuronal synapses may be different.

Recreational drugs come in all shapes and sizes and many have different mechanisms of actions, and responses that can depend on a number of factors such as a person’s physiology, drug dose and frequency of administration (e.g., acute versus chronic (repeated)), and brain region affected. In humans levels of many neurotransmitters in the brain are often reflected by the way that they bind to their corresponding receptors, e.g., by using techniques such as PET scanning. If we look at something like cannabis, an active ingredient such as tetrahydrocannibol (THC) acutely activates CB1 receptors to increase dopamine levels in a reward area like the striatum (e.g., see - http://www.ncbi.nlm.nih.gov/pubmed/25801289) but ‘chronic cannabis abuse’ may be associated with decreases or no change in the concentration of this neurotransmitter in the same brain region. I’ve seen data where ‘actual’ striatal dopamine levels have been increased by 25-100% by acute THC administration in rats, or by up to around 150% in humans (e.g., see - http://www.nature.com/npp/journal/v34/n … 138a.html) which may on occasions actually be lower than those obtained playing a video game where dopamine levels in the (human!) striatum may increase two-fold (e.g., see - http://www.nature.com/nature/journal/v3 … 6a0.html).

Even if we had accurate measurements of brain neurotransmitters in your comparisons, it is what these chemicals do functionally that is important - e.g., there are drug-receptor theories about occupancy where functions plateau at certain neurotransmitter levels (‘more’ does not necessarily give ‘bigger’ or ‘better’ responses), and after repeated administration many responses desensitise such that ‘more’ is required to give the ‘same’ response.

Some species of fleas do not mind the cold (although not freezing!) so much - see http://onlinelibrary.wiley.com/doi/10.1 … 374.x/epdf

There are a number of comments about fleas on-line that quote “C.felis has extended its range from into northern temperate climates despite its inability to survive exposures to temperature below 30.2 F (-1 C) for more than five days in any life stage”. The article where this statement is taken can be downloaded from: http://www.researchgate.net/publication … a_Problems

The original reference is: Silverman J, Rust MK. Some abiotic factors affecting the survival of the cat flea, Ctenocephalides felis (Siphonaptera: Pulicidae). Environ. Entomol. 12: 490-495, 1983.

Here is a very good (but lengthy!) overview on DNA sequencing by (fluorescence-based) capillary electrophoresis - https://www3.appliedbiosystems.com/cms/ … 041003.pdf

The first 20 or so pages give an excellent overview of the method, one of the technical advances in ‘high throughput’ DNA sequencing that led to the elucidation of more DNA sequences at a faster rate. Note that this and other DNA sequencing methods are often referred to as ‘automatic sequencing’ - by and large though all require manual intervention at some stage, e.g., in the sequencing template preparation, loading gels. Also note that capillary DNA sequencing is not restricted to plasmid DNA templates - e.g., genomic DNA that may or may not be expressed in other vectors (such as BAC) is often sequenced. In addition the primers are not always directed to a vector sequence, as Ajna indicates - you can 'walk' a DNA sequence by designing specific primers as you go along.

Good question, Lucy. Very simply, if you filter something out of the blood you would want to re-absorb the ‘good stuff’ back into the circulation. I showed your post to colleague Dr Mark Knepper (from the National Institutes of Health in the States) who is an expert on kidney physiology and has done seminal work on renal transport mechanisms - his response is as follows:

“The glomerular barrier filters large molecules like proteins, but not small molecules like glucose or ions like phosphate. So, the glomerular filtrate at the beginning of the proximal tubule has basically the same composition as blood plasma except for most of the protein molecules. Valuable molecules and ions like glucose and phosphate must be actively transported back to the blood by the proximal tubule. The plasma membrane of proximal tubule cells contains specialized transporters, e.g. SGLT2 for glucose and Slc34a1 for phosphate, that carry out this function. There are other substances that are ‘reabsorbed’ via other specialized transporters. Interestingly, a new therapeutic approach to diabetes mellitus is to inhibit the transport function of SGLT2 using drugs like canagliflozin, dapagliflozin and empagliflozin.

So, why didn’t the evolutionary process just make the glomerular filter tighter to small substances and not have a proximal tubule to reabsorb all those substances? This is an unanswered question, but one can speculate. The existence of aglomerular fish (Goosefish) that have proximal tubules but no glomeruli raises the possibility that the proximal tubule was there first. In these fish, substances get into the urine by active transport into the pro-urine (tubular secretion).  One idea then is that there was co-evolution of the glomerulus and proximal tubule allowing proximal tubules to acquire reabsorptive functions along with the development of glomeruli.”

see also a previous post - http://www.askabiologist.org.uk/answers … hp?id=7500 which talks about the filtrate and urea excretion

The vast bulk of studies in this area have been performed in E. coli. As David suggests, E. coli counterparts to L-arabinose operons also appear to be present in other bacteria; see - http://www.ncbi.nlm.nih.gov/pubmed/9084180, and some lactobacilli apparently possess the gene (AraA) for catalytically-active L-arabinose isomerase (http://www.ncbi.nlm.nih.gov/pubmed/18031349). In addition, for another gene in the operon, AraC (regulating gene), there appears to be quite a few homologues (some with only a few amino acid differences) that regulate arabinose-catabolizing genes in response to arabinose in other bacteria - see Table 1 in http://gene.bio.jhu.edu/Ourspdf/127.pdf

Alternative polyadenylation can be due to alternative polyadenylation sites (i.e., more than one possible site) found within the same terminal exon, or can be located within distinct exons. mRNAs within this latter group can include exons that have been alternatively spliced - that is, in alternative splicing when: (1) one or more exons (with polyadenylation sites) has been skipped polyadenylation may take place in another unspliced terminal exon, or (2) a 5' splice site has been skipped polyadenylation may occur downstream in an otherwise intron. The existence of alternative splicing and multiple possible polyadenylation sites are ways in which transcriptional diversity (one gene often gives rise to more than one mRNA product) is achieved.

Here is an article (see Fig.1 for summary) that goes into alternative polyadenylation in detail - http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3504663/

(posted in Genes, Genetics and DNA)

I think the capacity for liver ‘regeneration’ as a method of compensatory growth is common to all vertebrates, which makes sense since the organ is critical for detoxifying chemicals and carbohydrate metabolism. From what I have read fish livers, in contrast to their mammalian counterparts, actually undergo authentic regeneration - i.e., restoration of original hepatic shape and size.

In many invertebrates the equivalent tissue of a liver is found, sometimes as outpouchings of the stomach or gut, where it has functions that include nutrient absorption and storage. As to ‘regenerative’ capacity I don’t know, but I found the odd abstract/article on the regeneration of the snail and crayfish ‘hepatopancreas’ - http://www.fasebj.org/cgi/content/meeti … acts/lb401 and http://link.springer.com/article/10.1007/BF00201646. I wouldn’t be surprised if re-growth is a fundamental characteristic of many hepatic-like tissues.

Here is a good review on the mechanisms of liver regeneration - http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2701258/

See also previous link - http://www.askabiologist.org.uk/answers … hp?id=4065

I can see your quandary! I think your quote from the Wikipedia link on ‘ribonuclease’ is referring mainly to eukaryotes, where polyadenylation in nucleus-encoded mRNA molecules is important for their stability - long poly(A) tails are generally stabilizing, and deadenylation is a prerequisite for degradation and usually occurs in the cytoplasm by dedicated exonucleases (endonucleolytic decay appears to be less important). So in your cells the first step is protection as 3’ poly(A) tails are complexed with poly(A)-binding proteins to regulate accessibility to exonucleolytic machinery.

In prokaryotes such as E. coli that lack a nuclear membrane, mRNA synthesis and degradation take place in the same compartment, and polyadenylation stimulates RNA degradation by endo- and exoribonucleases.

I refer you to this quote - “unlike in eukaryotic cells, only a small fraction of mRNAs is measurably polyadenylated at a given time in wild-type E. coli cells. We now know that this scarcity does not mean that polyadenylation is rare, but simply that the relative activities of PAP and of exonucleases that remove poly(A) tails in the cell are such that poly(A) tail length is kept to a minimum. Whereas the existence of bacterial polyadenylation is no longer disputed, the metabolism of poly(A) tails and, even more so, their biological role - particularly their impact on mRNA stability - appears at almost complete variance in eubacteria as compared to eukaryotes.” from http://www.sciencedirect.com/science/ar … 7402011376

Not trivial experiments, but it is easy enough to google something like ‘antiparallel versus parallel triple helix stability’ to retrieve examples. You are likely going to have to have oligonucleotides synthesised, and equipment to perform melting temperatures/profiles (e.g., different temperatures and ion concentrations), absorbance values/spectra (the latter requiring specialised equipment that most standard molecular biology labs would not possess), and gel electrophoresis.

see - http://www.ncbi.nlm.nih.gov/pubmed/11790150 and http://www.ncbi.nlm.nih.gov/pmc/articles/PMC308398/ as examples

(posted in General Biology)

Try -
dendrite
insipidus (as in diabetes insipidus)
adrenal/adrenaline
electrophoresis
flocculate (not regularly used but I like the word!)

The bacteriocidal action of antibiotics is concentration-dependent, but at the concentrations of Amp used in the lab (e.g., 50-100ug/ml) evidence of bacterial death is observed within hours, although a very small proportion of bacteria can be Amp-resistant (e.g., on agar plates you may observe Amp-resistant bacterial satellites surrounding colonies).

Excellent question(s) to which I do not know the answers, but I draw your attention to one of the few studies on this subject - http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3001074 (which it appears you may have read) and the following statements therein: “From a structural perspective RNA might appear to be an unlikely surrogate substrate for restriction enzymes, as the presence of a 2?-hydroxyl group adjacent to the scissile phosphodiester linkage can present a steric barrier to assembly of a catalytically competent complex with an enzyme that has evolved to bind and hydrolyze a substrate that lacks such a group (16). Also, crystallographic analyses of RNA–RNA homoduplexes and RNA–DNA heteroduplexes show that the presence of the 2?-hydroxyl group causes both to adopt an A-form helical structure characterized by an expanded minor groove and contracted major groove relative to canonical B-form DNA (18,19). As sequence-specific recognition by Type II REases commonly involves precise contacts between amino acid side chains and the edges of nucleotide bases in the major groove, such interactions are likely to be unavailable, at least in part, to an equivalent RNA substrate (20).”

Note that the cleavage of RNA in DNA-RNA hybrids described in this and other articles (e.g., see - http://www.ncbi.nlm.nih.gov/pmc/articles/PMC324136/) does not appear to be as efficient as cleavage of DNA-DNA duplexes. Further X-ray crystallographic studies may shed some light on the structural requirements (e.g., phosphate group and sugar modifications) for RNA cleavage by the ‘promiscuous’ restriction enzymes noted in the cited references.

A cDNA in a bacterial plasmid in the ‘sense’ orientation can be inserted between incorporated RNA polymerase promoter (upstream) and perhaps terminator (that may include a polyadenylation site) sequences - the RNA pol promoter would usually be used in this scenerio to transcribe synthetic RNA off the plasmid cDNA in vitro. The cDNA can be orientated by a variety of methods such as using linkers. For example, a good (and quite elaborate) example of this technique is described by Okayama and Berg (1982) - the original pBR322-based expression vectors (e.g., pCD1, pCD2) were later engineered to incorporate the SP6 RNA promoter. Simply, the 3’ end is orientated by virtue of the mRNA polyA-tail, while the 5’ end of the cDNA insert is tailed with oligo-dC to be annealed with an oligo-dG-linked plasmid vector. See Fig 2 in - http://berg-emeritusprofessor.stanford. … 161-70.pdf

If you are thinking about endogenous bacterial RNA polymerase activity I suggest that you google something to the effect of 'plasmid DNA replication in bacteria'.

Not only may the intradermal injection itself provoke an immune response, the intradermal injection of substances like vitamins may alter the function of the skin immune microenvironment, possibly affecting immune-competentancy via dendritic cells such as Langerhans cells (macrophage-like), and other recruited circulating or resident immune cells, e.g., see -http://www.tandfonline.com/doi/pdf/10.4161/hv.22918

I agree with David in that it would probably take large amounts of carotene. I've certainly heard of people attributing an orangy-dark brown hue in dog hair to  carotene in the diet. Obviously this would all be happening on a background of coat colour genes and I think hair colour in ferrets is quite dynamic depending on physiology (e.g., reproductive state).

G'Day Izabele: In short, just as different ‘foods’ can provide varying nutritional value to many animals, different media compositions (e.g., a variety of types of agar with nutrient supplements such as glucose, salts, vitamins, amino acids and nitrogen, combinations of which are usually essential for maximum growth) can support different colony growth (or none at all) depending on the bacteria’s metabolism. Some bacteria are more fastidious than others! The (lack of) moisture content of the media can also affect growth.

(posted in Evolution)

Here is another example of what Alistair mentions - http://www.pnas.org/content/109/8/2966.full.pdf

It points to some ancestral enzymes evolving (e.g., through multiple duplication and other events) from low to high activity.

(posted in General Biology)

There is a lot of experimental data on 'what connects to what' (‘connectomes’) but one problem is assimilating all the information into concise, user-friendly formats. This is being addressed in many labs, and we should see many more neural connections revealed with the current popularity of optogenetics combined with pharmacology. As David says, it is currently quite difficult to accuratley define a 'network', and we know that the components of many neural pathways overlap. Curation of ‘historic’ neuroanatomical literature (over 16,000 studies on defined axonal connections between rat cortical regions) gives you an idea of a small part of the incredible complexity. For example, see original article - http://www.pnas.org/content/early/2015/ … 2.full.pdf - and commentaries - http://neurosciencenews.com/cerebral-co … tome-1931/ and https://news.usc.edu/79313/study-reveal … of-rats/).

G'Day: Going on your post heading first, the PD bond is a strong covalent bond, as are most 'peptide bonds'. The strength of the covalent bond depends on the distance that separates the 2 nuclei, so it varies depending on the atoms involved. As chemical bonds go, covalent bonds are relatively resistant to heat denaturation, and in the case of DNA and proteins the 'first things to go' will be the weaker hydrogen and other bonding.

I would guess that in general DNA is more heat-resistant (chemically rather than necessarily functionally) than most proteins because a significant amount of the ‘other’ bonding in proteins (depending on their structure!) is hydrophobic (also present in DNA; in proteins it helps maintain complex folding as in tertiary-quaternary structures) which is even weaker than hydrogen bonding. However some proteins are very thermostable outside the normal physiological range (an example of this in the lab is taq polymerase) - one reason for this apparently is the presence of extra hydrogen bonds (see Wikipedia entry on protein thermostability).

(posted in Genes, Genetics and DNA)

G'Day Niki:There are quite a few ‘Ligation Calculators’ (just google this term) available on-line, where you can enter your vector/insert information, e.g.,
http://www.insilico.uni-duesseldorf.de/Lig_Input.html
http://www.genelink.com/tools/gl-lc.asp
http://www.promega.com/a/apps/biomath/i … c=molarity

If you are working with pmol ends, e.g.,

for dsDNA:

~MW = 700 Daltons/bp (a more accurate figure is 649 Daltons/bp)
(e.g., MW 1kb is ~7 x 105 daltons)

Therefore, ug in pmole = length x 649g/mole x 10-12moles/pmole x 10-6ug/g
                                  = length x 6.49 x 10-4mg/pmole
                                     (x2 for pmoles 5’ ends)

e.g., 1kb DNA = 1000 x 6.49 x 10-4 ug/pmole = 0.649ug/pmole
: 1/0.649 pmoes = 1ug
: 1.54 x2 (for 5’ ends) = 1ug

e.g., pGEM4Z = 2746 x 6.49 x 10-4ug/pmole
= 1.781ug/pmole

: 1/1.7821pmoles = 1ug
: 0.56 (x2) = 1.122 pmoles = 1ug

You may have to vary your vector-insert ratios (e.g., lower and higher than 1:3) to obtain optimal ligation efficiencies - in the end you probably only need a few transformed bacterial colonies to obtain a successful ligation!

As part of nervous system disorders chemokines are produced by a number of cells including activated astrocytes and microglia. But as David says they (and their receptors which are mostly G protein-coupled) are constitutively expressed by glia and neurons and may act as neurohormones/neurotransmitters particularly in neuroendocrine systems to modulate our water balance, feeding, body temperature and response to stress. There a quite a few reviews on this such as:

"Chemokines and chemokine receptors: new actors in neuroendocrine regulations" by Rosten, W et al. Front Neuroendocrinol 32: 10-24, 2011.

Note that there are people with conditions such as hyperacusis or micophonia who have decreased tolerance to, or 'hatred' of what many of us would consider rather innocuous sounds, such as pen-clicking, that may have behavioural consequences (e.g., see - http://www.ncbi.nlm.nih.gov/pubmed/25726280).

(posted in Genes, Genetics and DNA)

The simplest RFLPs are caused by single base pair substitutions, or single nucleotide polymorphisms (SNPs), which occur more frequently within non-coding elements such as introns or intergenic sequences. While complex rearrangements such as insertions, deletions, duplications and translocations are also most often found within non-coding DNA (however note that recent estimates are that about 80% of our genome ‘does something’, and it does not necessarily have to be protein-coding to be functional; e.g., see - http://www.nature.com/nature/journal/v4 … 247.html), restriction enzymes generate DNA size differences in RFLP analyses by cutting both and/or either coding and non-coding DNA, so as David says RFLPs are generally markers that are distributed throughout the genome.

See also Fig 8.3 in - http://www.informatics.jax.org/silver/c … /8-2.shtml

Non-digestion by a restriction enzyme is a typical cause - partials would likely give you more bands than you expect. The level of star activity depends on the enzyme you used (HindIII is what we would call a 'good or easy' cutter compared to some other enzymes) and can also give more bands than expected. Non-digestion, partials and star activity can have many causes, including too much DNA in the digest and/or too little or excess enzyme added; 'impurities' in, or incorrect buffer constituents; methylation of a restriction cleavage sites. The troubleshooting guides of some restriction enzyme vendors can sometimes be useful in identifying potential problems. Make sure you have calculated the theoretical band sizes correctly!

Measuring beta-Gal activity, as Reetika suggests, will allow you to screen a large number of bacteria to determine whether they are expressing the plasmid (but may also detect a number of false-positives, albeit likely very small for this transformation). Electroporation and calcium phosphate precipitation are two methods of introducing the plasmid (the transformation) into the bacteria; calcium chloride is one chemical agent that is used to make some bacterial strains more competent (take up more DNA). Heat shock is used to enhance the plasmid DNA uptake. The amp is used to select for bacteria that have taken up the plasmid (expressing the amp gene).

You could also test for transformation effeciency by other methods that would determine whether plasmid DNA was expressed in the bacteria, e.g., PCR, colony hybridisation using radiolabelled probes to the pUC8 DNA sequence but these involve a little more work.

So-called conservative amino acid substitutions replace an amino acid with another amino acid of similar chemical structure (e.g., charge). For example, serine may be substituted by another polar, neutral amino acid such as threonine - both are notable as potential phosphorylation residues within proteins -  which may not (or may!) affect a protein's function. If you are interested in amino acid properties (and possible substitutions) see a reference like - https://biokamikazi.files.wordpress.com … utions.pdf

(posted in Plants & Fungi)

Plants do not have sensory organs and the scientific community consensus is that they do not have 'feelings'. Backster's experiments have been widely discredited - if you want more information google 'Backster plant experiments'.

I agree with David - novel environments cause anxiety in many species, often activating the hypothalamic-pituitary-adrenal 'stress' axis to release corticosterone in addition to altering behaviour.

Antarctic icefish don't make haemoglobin, and pump a lot more blood than other fish to compensate. They may have other adaptations. I think they also don't have myoglobin in some muscles!

(posted in General Biology)

There is a tendency for people to use the term ‘monovalent’ slightly differently. Antibodies are ‘bivalent’ in nature, having two antigen binding sites due to the Fab (fragment, antigen-binding) portion of the molecule which is composed of one constant and one variable domain for each heavy and light chain of the antibody. This is not to be confused with antibody molecules that act as monomers (e.g., IgG, IgE), dimers (IgA) or pentamers (IgM).

Strictly speaking a monovalent antibody has only a single binding site for an antigen (as distinct from natural ‘bivalent’ antibodies), i.e., a composed of a single antigen-binding arm. An IgG antibody digested with the enzyme papain is monovalent with two Fab fragments of about 50 kDa in size and an Fc fragment. In contrast IgG digested with the enzyme pepsin removes most of the Fc fragment but leaves the Fab portion intact, yielding two interconnected antigen-binding portions that are divalent.

The term monovalent is also used to describe an antibody that has affinity for only one epitope or antigen, or a single type of antibody. So monoclonal antibodies are often termed monovalent (compromised of one type of ‘bivalent’ antibody) for the same epitope.

I don’t want to confuse you, but various configurations of antibodies that may be therapeutically useful (e.g., altering serum half-lives and/or tissue penetrability) can be molecularly engineered. So in terms of ‘valency’, e.g., you can have an engineered antibody that is bispecific, binding one antigen in a bivalent and another antigen in a monovalent manner, or an antibody binding different antigens simultaneously in a monovalent fashion! (e.g., see Fig 1 in - http://www.sciencedirect.com/science/ar … 1400178X).

This is a very interesting topic and you will find a literature search will retrieve quite a few research articles. For example, there is the general idea that ‘magnetotactic’ bacteria, commonly found in the mud of marine environments, have organelles containing magnetic crystals in the form of iron compounds (magnetite or iron sulfide) that aid them in reaching (navigating to) regions of optimal oxygen concentration (see -http://en.wikipedia.org/wiki/Magnetotactic_bacteria and http://blogs.scientificamerican.com/lab … acteria/). There is also a study (see commentary at -http://www.nature.com/news/2004/041126/full/news041122-13.html) in bacteria that have been engineered to lack a carotenoid (which usually absorbs oxygen free radicals) where a magnetic field might affect their growth. In gram-negative bacteria it has been proposed that low-frequency magnetic fields retards their growth, affecting both viability and oxidoreductive activity (see - http://www.ncbi.nlm.nih.gov/pubmed/11786365).

Re Mike's mention of cats 'fishing' there was a TV program ("Africa's Fishing Leopards") on recently where a leopard and its two cubs developed a technique to catch catfish in a river with low water.

This is not a topic I know much about but it is an interesting question. I think it could be quite difficult to obtain samples at the same stage of development, or without plasticity changes due to environmental/social factors, and the brain structure of fish varies quite considerably across species. I also would imagine that there is considerable debate as to how encephalization and ‘intelligence’ might be related! I guess the EQ at least takes into account divergent body sizes across different types of fish. Rays are said to have the highest EQ amongst fish but sharks also rate highly, at least compared to tuna! see - https://domainmonkey.wordpress.com/2012 … -animals/; http://bio.sunyorange.edu/updated2/crea … brain.htm. If the EQs ranges quoted in these articles are correct (0.25 to 2.77) that would place some sharks at the higher end above many modern reptiles and small mammals.

FYI, here is a link about the sizes of shark brains - http://www.elasmo-research.org/educatio … brain.htm, and a paper on fish brain size evolution - http://www.sciencedirect.com/science/ar … 2212013863

Variations in the shade of green are influenced by a number of factors (see the link in Reetika's post), especially the amount, type and distribution of chlorophylls (of which there are 6 different, structurally similar types, alpha and beta being the main pigments, that absorb blue and red light and hardly any green light which is reflected back to our eyes) and other pigments such as carotinoids (light reflected appears yellow or yellow-orange) and anthocyanins (leaves appear red or purple to us) that can blend or even mask the green pigments.

I think that quite a few natural pigments require enzymes and substrates for biosynthesis so appropriately expressing all of these components in bacteria may be problematic. You can certainly bacterially express and manipulate light-sensitive pigments such as luciferase (which is already endogenous to some bacteria) and phytochromes.

As Reetika says, the immune systems of animals are adaptations to their environment and behaviour. In terms of immune system ‘strength’ the antimicrobial activities of serum from American alligators (often living in territory teeming with bacteria and other microbes) are very impressive (see - http://www.ncbi.nlm.nih.gov/pubmed/16298430 and commentary at http://news.nationalgeographic.com/news … blood.html which highlights the apparent broader range antimicrobial activity of alligator versus human serum). Sharks and bats also have very effective innate immune systems.

G'Day Adrian: Comparing the sequences of 2 genomes can be done with programs that perform ‘whole genome alignments’ (WGAs), or compare the DNA sequence of individual genes or chromosomal regions (often using the BLAST alignment program at NCBI - http://www.ncbi.nlm.nih.gov/, or BLAST-like software). Sometimes only protein-coding genes are compared. The early figures of around 98-99 percent of DNA sequence identity between humans and chimps were not based on comparisons of the entire genomes of both species - the data is usually presented as such, e.g., 98 percent identity over a coverage of X percent of the total genome which may include gaps for inserted/deleted sequences (if these are included the percent difference will increase), with Y amount of quality (sequencing accuracy).

There are likely DNA sequences common to various viral family genomes (which vary considerably in size) but I think it is quite unlikely that there will be a 4-8bp non-repeat sequence common to all, or even most DNA viruses, and that are not found in any other organisms. There are about 4,500 complete viral genomes (of which approx. 1/3 are RNA viruses) deposited in the NCBI database. Unless there was a batch entry format in BLAST where ALL viral sequences could be compared to one another at the same time (and not a single viral genome entry screened against all available viral sequences) it would not be a trivial task to identify a specific 4-8bp sequence, even if it did exist!

see also a previous link on cat taste receptors - http://www.askabiologist.org.uk/answers … p?id=10898 and http://jn.nutrition.org/content/136/7/1932S.full (that says cat Tas1r2 is a pseudogene (non-functional); the Tas1r2 and Tas1r3 heterodimer encodes the sweet taste receptor in mammals). As far as I can make out cats do have the genes encoding bitter taste receptors (the Tas2r family; see - http://www.ncbi.nlm.nih.gov/gene/?term=cat+tas2r).

Optical imaging of intrinsic signals relies on illuminating the area of interest with different light wavelengths depending on the source of the intrinsic signal. The signals can include changes in blood flow/volume as you mention and activity-dependent changes as outlined in the Introduction to your reference.

As described in: http://www.sciencedirect.com/science/article/pii/S0165027004000974, illuminating light wavelengths are manipulated with filters such as green (better to see blood vessels), orange, red and near-infrared. The images of reflected light are recorded with a CCD camera.

Besides strictly evolutionary perspectives, there are a number of studies that have tried to address the biological basis for some of the examples you list. For music rhythm there is this idea that there is a relationship between music and speech rhythm (see - http://www.ncbi.nlm.nih.gov/pubmed/25536848) and that brain circuitry for complex vocal learning is required to perceive (http://www.mdpi.com/2076-3425/4/2/428) and follow a beat (present in parrots! see - http://www.ncbi.nlm.nih.gov/pubmed/19409786). The cerebellum has also been associated with musical rhythm and finger tapping (see - http://www.ncbi.nlm.nih.gov/pubmed/25583606). As to our response to cat purring, there are suggestions that cats may be exploiting the innate tendencies that humans have for nurturing offspring (the peak frequency of cat purrs being similar to that of a baby’s cry); see - http://www.sciencedirect.com/science/article/pii/S0960982209011683.

As an aside, lots of what we do occurs in patterns - we all have various types of endogenous biological rhythms, e.g., sleep/wake cycle, circadian corticosterone and the release of other hormones/neurotransmitters, brain theta oscillations, etc., many of which are centrally co-ordinated by the ‘master clock pacemaker’ in the hypothalamus, the suprachiasmatic nucleus. More or less independent oscillators (e.g., ‘clock’ genes) that can be influenced by the brain are also found in peripheral tissues such as the liver, skeletal muscle, adrenal, pancreas, heart and testis.

G'Day Sammy: There are whole range of optical techniques for imaging the functional activity of the brain in vivo that do not rely principally on blood flow. These have been used in the vast majority of cases in animal studies, and include those that utilise voltage-sensitive dyes, 2- and 3-photon imaging, intrinsic signal optical imaging (based on when the brain is illuminated neuronal activity causes changes in the intensity of light that is reflected) and fluorescent genetically-encoded probes/sensors for molecules like calcium and ATP (e.g., see - http://www.nature.com/nmeth/journal/v11 … .2773.html and http://pubs.acs.org/doi/abs/10.1021/ac4015325) which are becoming more and more popular as biosensors are optimised.

Such techniques vary in their level of temporal (e.g., milliseconds) and spatial (microns to millimetres) resolution, use of signals intrinsic to the brain or external probes, degree of brain invasiveness and affordability (requiring specialised equipment).

An obvious advantage of the measurement of blood oxygenation level dependent (BOLD) signal in functional MRI, that is dependent on blood flow, is that this non-optical technique for measuring neural activity is non-invasive (and has been adapted in some situations for awake subjects). fMRI-BOLD also measures generalised activity rather than a specific biochemical process (e.g., calcium signalling) within the brain or most, if not all processes (e.g., ATP).

Another way to check your DNA concentration is to do serial dilutions of your DNA and control DNA in your DNA-staining solution (e.g., ethidium bromide), and spot them on some plastic or cling-film and view under UV light. It will give you an idea whether the DNA (or RNA!) is present (but not whether it is degraded) before you run it on the agarose gel.

Just to briefly add to David’s comments, and continuing on from the wikipedia page, there is a lovely paper from early 2014 that showed in studies in mice that the patterns of X chromosome inactivation varied widely from tissue to tissue and sometimes showed distinct left-right asymmetry - see http://www.ncbi.nlm.nih.gov/pubmed/24411735.

While there are inherent difficulties with the microbe-based approach, and although the paper I refer does not exactly follow the idea Billie presented, the claim is that glucagon-like peptide-1 (1-37) derived from bacteria stimulates the conversion of both rat and human intestinal epithelial cells into insulin-secreting cells (that are glucose-responsive to some degree). I think this is an interesting idea, especially if the method can be fine-tuned and these re-programmed intestinal cells can maintain regulated insulin production indefinitely.

G’Day Billee: This type of idea has been tried for tumors (i.e., directly injected bacteria expressing an engineered gene). It has also very recently been reported to have been used in a model of diabetes in rats - see the abstract entitled “Engineered Commensal Bacteria Reprogram Intestinal Cells Into Glucose-Responsive Insulin-Secreting Cells for the Treatment of Diabetes” at http://diabetes.diabetesjournals.org/co … hort?rss=1 (unfortunately I can’t access the journal). There have been a lot of commentaries about this in the press in the press.
Potential problems with your scenerio(s) include: it would likely have to be tested in animals first and if successful there is still no guarantee that it would work in humans; immune mechanisms reacting to the introduced bacteria causing sickness and/or bacterial death; constitutive induction of your promoter driving gene expression (insulin in your example); change in the bacterial genotype and/or phenotype over time.

(posted in Evolution)

Neuropeptides act as hormones in the classical endocrine sense, neurohormones (acting at some distance from where they are released from neuronal cells in the brain) and neurotransmitters (acting at neuronal synapses). For the evolution of neuropeptide signalling, e.g., see - http://www.sciencedirect.com/science/ar … 512000687.

The receptors at which they act, mainly G protein-coupled receptors (GPCRs), have also co-evolved with their ligands in a number of cases (e.g., see - http://www.ncbi.nlm.nih.gov/pubmed/20708652; http://www.els.net/WileyCDA/ElsArticle/ … 150.html).

Adrenaline also acts at a subfamily of GPCRs as a aminergic hormone and neurotransmitter. See comments in a previous link - http://www.askabiologist.org.uk/answers … p?id=7082. The book chapter “Evolutionary considerations of neurotransmitters in microbial, plant, and animal cells” by Roshchina VV (2010) gives a reasonable overview of neurotransmitter evolution, and see, e.g., - http://www.nature.com/nrn/journal/v10/n … n2717.html for a review of synapse and neurotransmission evolution.

I would add that studies on the evolution of hormones and neurotransmitters, and their receptors, have been aided by the sequencing of a number of vertebrate and invertebrate genomes.

Discounting any other sort of artifact I think sugar crystal is a good shout (salt crystals like to form cubes) for something on a slide, although it looks a bit too regular and transluscent even for a crystal hexagon, if indeed that is the shape (I can't tell from this magnification with my eyesight!). If you focus in-and-out do you get the impression of a 3D structure? Was there only one of these shapes in your prep?

There are so many things that could be going on here! Are you using a column-based method for plasmid DNA purification? Are all of the componenets of the kit working optimally (e.g., can you isolate decent yields of another plasmid)? I have no experience growing up plant expression vectors (and pCAMBIA 1304 is quite a large one) but for some difficult-to-grow mammalian expression plasmid vectors LB Super Broth can help. In extreme (consistently bad DNA yields) cases I have even plated out plasmid-containing bacteria on large LB agarose petri dishes, rather than in liquid LB broth, and scrapped the bacteria into the initial plasmid lysis solution. Perhaps it would be a good idea to have a chat about this with your PhD supervisor...

G'Day Nick: Some B cell antibody responses do not, in fact, require T cell help, e.g., T cell-independent antigens such as some polysaccharides in bacteria can activate B cells directly. Other antigens require T-B cell collaboration, including many that require processing, perhaps packaging, and presentation to B cells, which may (I imagine not always, but I have no idea of the time-courses) take a bit longer than direct activation of B cells. There are many simple and detailed articles available on this, e.g., have a look at http://www.ncbi.nlm.nih.gov/books/NBK27142/ for one description. The Wikipedia entries on T cell-dependent and -independent activation may also help. The nature of the antigen is important, along with many other facets of the immune system including cooperation of other antigen presenting cells that may secrete immune cell-modifying molecules like cytokines, immune microenvironment, host status, etc.

[Note that there are T-helper cell subtypes that are functionally different (e.g., for a simple summary see - http://www.uptodate.com/contents/t-helper-subsets-differentiation-and-role-in-disease]