biochemistry - Can siRNA induce DNA methylation in mammalian cells?

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

In plants, dsRNAs targeted to CpG islands within a promoter can also
induce RNA-directed DNA methylation^{3, 4, 5, 6, 7, 8}; however, it
remains unclear whether gene silencing mediated by DNA methylation can
be induced by dsRNAs in mammalian cells. Here, we demonstrate that
short interfering RNAs (siRNAs; 21–25-nucleotide RNA molecules) induce
DNA methylation and histone H3 methylation in human cells.

This paper seemed to show that siRNA can also induce long-term silencing of genes by DNA methylation in mammalian cells, which hasn't previously been observed. Unfortunately, the paper was retracted later, calling the results into question.

I found the paper very exciting at the time, as it suggested the possibility to perform gene therapy, to silence specficic genes, just by using RNA interference.

What is the current scientific consensus here, is there any convincing evidence that siRNA-induced DNA methylation is possible in mammalian cells? Was anyone able to replicate the results from Kawasaki and Taira?

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]

synthetic biology - DNA synthesis companies: cost per base, turn-around time, codon-optimization algorithms

Are you working in academia? It might be worthwhile to call the companies directly and see if there is a discounted rate for your institution (another lab or department may have already set something up).

You won't be able to get information on the different algorithms if they are proprietary. However considering that codon usage data is public and shared knowledge, I can't imagine that the different algorithms are that much different. I think there are some tricks for choosing certain codons near the transcription start site in some organisms? From what I hear, the algorithms are constantly changing. I could tell you how my E. coli optimized gene from GenScript worked two years ago, but they'd probably design it differently now.

Okay - GenScript calls their algorithm "proprietary", so they are not sharing the details. DNA2.0 says their algorithm is patented, so it's out there for inspection. They published a PLOS paper on their E. coli algorithm: Welch et al..

Although, maybe all they do these days is spit out codon sequence randomly:

The results obtained in this study indicate that the codon
randomization method is a superior strategy for codon optimization. A
significant improvement in protein expression was obtained for the
largely established process of chymosin production, showing the power
of this strategy to reduce production costs of industrial enzymes in
microbial hosts. (Menzella 2011)

Stitching smaller synthesized fragments works well if you're pressed for time, but it's usually more expensive. However, sometimes these companies do special pricing on synthesis for <1kb, which makes stitching a great choice. Turnaround is generally faster for three 1kb fragments than one 3kb fragment.

bacteriology - Are the intestinal microbiota substantially different within a diverticulum of the colon?

The appendix is a type of true diverticulum of the colon, although it is one that is present in everyone (unless a surgeon has removed it). There are bacteria in the gut from the mouth all the way down to the anus, and the numbers and types of bacteria change along the length of the intestinal tract. A recent review from J. Ridlon published in the Journal of Lipid Research (2006) is available online and has documented these observations from a number of investigators. In that paper the bacterial species are described as changing as well as the relative abundances of bacteria which increases along the length of the intestinal tract. Figure 1 from that paper which is open access available from the link above is below.

I think its interesting that the wiki page you're citing states that individuals without an appendix were more likely to develop a recurrent C. diff infection. One could interpret that as they did on that site that somehow the appendix is repopulating the colon and making that individual more resistant to a repeat or "Recurrent" infection. However, I don't know of any studies that have directly tested that hypothesis.

In terms of whether or not the gut flora - aka "gut microbiota" - are significantly different in the appendix is possible, but I don't think anyone has identified that just yet. One of the problems with this would be that we don't routinely remove appendices unless they are infected/inflamed which intrinsically would alter the microbiome locally.

So, is it possible that the microbiome could be significantly different in the appendix itself compared to the cecum just a few centimeters away? The answer would be - potentially, but no one has been able to address that question to date.

human biology - Why is rabies incurable?

This is because rabies is a viral infection of nervous tissue that propagates through peripheral nerves into the brain and causes brain tissue inflammation (encephalitis).

As long as the virus is in the brain there is no way to get rid of it. The main trade-off here is that everything that would kill the virus will be as (or even more) aggressive against the brain tissue, and impairment of the latter will lead to really heavy deficits in vital functions like breathing and thermoregulation.

The first manifestations of rabies are those due to brain damage. This means, the virus is already there and the brain is already fatally damaged.