biochemistry - What happens to dextrorotatory amino acids in humans?

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

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

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

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

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

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

An excellent review

• D-Amino Acid Oxidase: Physiological Role and Applications

  
  
  

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

  
  
  

  Biochemistry (Moscow) is freely available from here

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

Additional Reference

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

biochemistry - How to wash the column during protein purification with GST tag?

I have been working with GST tagged proteins for the last 4 years and after loading the cell lysate into the column I was washing it with 20-30 column volumes of PBS and sometimes my proteins were eluted very pure sometimes with a lot of impurities.

A few times I tried washing with NaCl gradient (0-400 mM) but later decided not to do that because it may denature the protein and some of my proteins may not refold.

What do you suggest for washing step?

Note: I don't have any UV detector to see the washouts/flowthroughs/elutions.

homework - Which enzyme catalyzes transcription and which translation?

Sorry, but you started on the right track. What you're looking for is called the central dogma of protein synthesis.

Genomic DNA is transcribed in the nucleus into messenger RNA (mRNA) by RNA polymerase. RNA polymerase is DNA-dependent; it needs a DNA template to make an RNA version of the message.

The messenger RNA moves into the cytoplasm where it gets translated by ribosomes into an a polypeptide (protein). Ribosomes are the enzyme that do the translating, and perhaps your confusion is because ribosomes are made up of both RNA molecules and proteins (a ribonucleotide complex).

Aminoacyl tRNA synthetases are the enzymes that make aminoacyl tRNAs (tRNA for short). These tRNA bring individual amino acids to the ribosome as the ribosome is extending the polypetide product of the mRNA message.

software - Statistical Analysis of Protein Folding Problem

One of the quickest ways to get oriented on what is going in the world of protein folding and modeling is to look at the proceedings of the Critical Assessment of Structure Prediction (CASP). CASP is basically a contest, held every 2 years where anyone can use their algorithm to predict the 3D structure of a protein whose structure is known, but not publicly available.

Its been a few years since I reviewed them results much - it looks like this year was interesting, but a perennial winner has been Rosetta, which has turned into an edifice of many suites of software which each execute different tasks in protein folding and modeling.

Open source software is pretty hard to find in this field. The software is complex. It usually includes components of machine and statistical learning, molecular dynamics, specialized algorithms that build up the protein one residue at a time, others which manipulate blocks of the protein structure around in space, electrostatic calculations, you name it. In addition, the software, once it gives some sort of result is quite valuable. I don't think any of these suites has really been released. I know that Rosetta is available to use as a web service, but you have to apply for access to the source. I don't think its an easy thing to get.

Some of the most complicated components are available open source. Molecular modeling and molecular dynamics open source software is quite sophisticated. I think we need an open source protein folding suite open source. I think David Shortle's algorithms might be a candidate for such a suite as its not so complicated and it works in some cases.

This field is pretty obscure and difficult to get around in. There aren't any easy introductions that I know of. Protein structures are computationally expensive and painful to work with in terms of writing software. On the other hand protein folding that really works is a revolutionary breakthrough, at least equal to the impact of the development of computers as a technology.

molecular genetics - How to calculate virus titre from qPCR

I harvested some lentivirus from 293T cells and want to titre the result. I infected 293T cells on a well plate with 400,000 cells per well which I infected with virus stock, and 1 in 10, 100 and 1000 dilutions (as well as a few uninfected wells). After 72h incubation I trypsinised the cells and used FACS to titre first, but 293T do not express the promoter under which GFP is located in some samples and hence these do not appear on FACS. As an alternative, I extracted the genomic DNA from all samples using a QIAGEN DNeasy kit (also the samples which I was able to titre by FACS already) and then performed qPCR on them.

I used the ABI PRISM 7000 SDS: http://www.cgenetool.com/products/abi_prism_7000.shtml

Included on the PCR plate were:

• Number standards: 1000, 10k, 100k, 1m copies


• Samples in triplicate


• No-template controls as well as untransduced controls

Each of those was doubled, once with primers and probe for WPRE (an element specific for the lentivirus) and once with primers and probe for beta-actin (a housekeeping gene present in all cells).

The results returned by the machine include for each well the cycle at which the threshold fluorescence was crossed - along with the automatically calculated quantity of copies (which the software calculates from the standards I assume).

Since I named the triplicate samples identically, it also automatically calculated the mean quantitiy among each triplicate (practical, eh?). I checked if there were any outliers which should have been excluded from the means but everything was fine.

So now for example I have a result of 700k for the quantity of WPRE in sample A. The quantity of beta-actin in sample A was 6.82 x 10⁶. The transfection was 400,000 cells per well, sample A was transfected with 10uL of viral stock diluted 1:100.

How do I calculate the virus titre per mL from this?

I have been given the formula: WPRE Qty / (b-act Qty*0.5) * cells in well (400k) * dilution factor (100), but am struggling to figure out a) why b-act Qty is divided by 2, b) whether the result would be virus titre per uL or per 10uL (transfection volume) and c) how beta-actin can have almost 10⁷ copy numbers consistently across all samples when there were only supposed to be about 10⁵ cells...

genetics - Can genetically modified genes jump to bacteria in the eater's intestine?

No.

There is nothing special about a piece of GM DNA when compared to any other random piece of DNA. If this phenomenon happened at any detectable level, we'd have found eukaryotic DNA in bacterial genomes/plasmids long before the introduction of genetically modified crops. And that would be front page news in the field of horizontal gene transfer! Which has many talented people in it!

statistics - Is it necessary to conduct a power analysis before beginning an experiment?

Due to my own woeful ignorance on the subject, I have been reading up on statistical methods recently. From what (little) I understand, the real answer to this question is:

Yes, but only if you are doing Neyman-Pearson hypothesis testing

and

Absolutely not, if you are using Fisher p-values

That is, the question isn't formulated correctly, because power analysis is only valid under one statistical framework (Neyman-Pearson). And you are probably not using that framework.

In my experience, most experimental biologists use Fisher's p-value, which gives the probability of the data (or more extreme data) assuming that the null hypothesis is true. Under Fisher's framework, among other drawbacks, there is no quantitative measure of the test's power. However, it has the benefit that it allows scientists to do something close to what we would like to do--that is to draw conclusions from evidence obtained in individual experiments.

The Neyman-Pearson framework does included the idea of a test's power, because you must formulate an alternative hypothesis as well as desired alpha and beta error rates before starting your experiment. However, it mostly denies us the ability to make inferences from individual experiments, and for that reason appears less suited to experimental science. To quote from Goodman (see below), under Neyman-Pearson, "we must abandon our ability to measure evidence, or judge truth, in an individual experiment."

There is no clear right frequentist framework, although what is clear is that you cannot mix Fisher and Neyman-Pearson. Finally, although it doesn't really address your question directly, it seems wrong not to mention Bayesian methods as an alternative to these two frequentist frameworks, which comes with its own baggage.

Further reading from people that understand this much better than me:

Michael Lew's answer to "Setting the threshold p-value as part of hypothesis generation" at Cross Validated

Michael Lew's answer to "What are common statistical sins" at Cross Validated

Hubbard, Raymond, and M. J Bayarri. “Confusion Over Measures of Evidence ( p’S) Versus Errors (α’S) in Classical Statistical Testing.” The American Statistician 57, no. 3 (August 2003): 171–178. (Working Paper PDF)

Arguments for Bayesian statistics:

Goodman, Steven N. “Toward Evidence-Based Medical Statistics. 1: The P Value Fallacy.” Annals of Internal Medicine 130, no. 12 (June 15, 1999): 995–1004.

Jaynes, E. T. Probability Theory: The Logic of Science (Online version of some parts)

lab techniques - Strange behavior of a DNA gel

I ran a PCR product of ~300 bp on a 2% TAE-agarose gel for 30 minutes. I used Sybr-safe as a DNA stain. Voltage was 80V.

When I imaged the gel, the DNA on the bottom half of the gel, including the ladder had disappeared (it showed no bands). The DNA in the top half looked-clear and well-separated, but the bands on the bottom were somehow missing.

What could be the reason? I've never encountered this before.

I know it's not due to gel overheating - I ran the gel in the cold room and when it was done it was cold to the touch.

genetics - Are identical twins exactly the same?

Errors in division occur all the time and can show up in any dividing cell; this is, of course, important for cancer biology. If one of my cells replicates oddly right now it likely won't matter since it's only one out of trillions, but if that happened at a very early age in development it could be present in many if not all of my cells. Identical twins indeed come from the same egg-sperm fusion event but they develop separately from a very early state. It's trivial to imagine a minor replication error in DNA at an early cell stage that results in different genotypes between so-called identical twins. Besides, we've known for years that identical twins can have major epigenetic differences, so this isn't particularly ground-breaking.

If we assume that identical twins are exactly identical, then if we make a clone of a twin, will all three be exactly identical?

Depends on your definition of "clone" but how identical is identical? If you mean exact, then probably not; at the very least they will have different numbers of cells, for example, not to even mention CNV or epigenetics. If you want some SciFi cloning thing then, well, join the club.

bacteriology - Is 1 g/l living biomass for a Biogas fermenter plausible?

A usual number for a healthy biogas fermenter is $10^9 - 10^{10}/mL$ Bacteria, 10-15% of which would be methanogenic archae. Exceptionally healthy fermenters have more total bacteria and up 25% archae.

The mass of E. coli is given with 670 femtograms. If E. coli is representative for fermenting bacteria that would put us on the order of magnitude of 1 g/L living biomass.

This seems like much to me, though this is just a gut feeling.
As additional information, such a fermenter will have a total solids content of about 10%. A typical loading rate will be 4-5 g VS/d l *, two thirds of which get metabolized to gas. To put it another way: The amount of digested biomass per day would be the same order of magnitude as the living biomass. However, as anaerobic digestion is a multistage process, with at least three steps, each population will have a higher rate of digestion to mass.

Are the numbers basically sound? Is E. coli an exceptional bacterium in terms of mass? Are archae generally as massive as bacteria? What are bacteria counts for other fermentation processes?

(*) g volatile solid per day and liter: for each liter fermenter volume, this many g potentially degradable matter are added per day

ecology - Does preservation in ethanol alter leaf litter mass?

I have benthic samples that were collected with an Ekman dredge from some small ponds. The samples contain quite a bit of coarse particulate organic matter (CPOM, basically dead leaves). I would like to quantify the amount of CPOM in the samples but they were preserved with 70% ethanol in the field.

Is there any evidence that ethanol preservation alters benthic CPOM mass?

ecology - In which month should I map maritime vegetation?

One way to approach this problem would be to look at herbarium collections of the taxa that you're interested in. Usually, a flowering or fruiting specimen is easiest to identify; but no matter what character is best for your taxon, if you look at the herbarium collections and see that
-most of the collections identified to species, or
-the especially good-looking collections, or
-flowering/fruiting collections,
were made in a certain month or season, that could help you to narrow down your time window.

As a bonus, in reviewing herbarium collections, you'll have given yourself a step towards being able to identify the species.

Some herbaria have some collections online; here, for instance, are records and images for online Puccinellia collections at a number of UK herbaria.