Guess who's BCKDK (this is BCKDK, or at least its structure). Image by Wikipedia user Emw.

Speaking of awkward acronyms: BCKDK.
It's branched-chain alpha-ketoacid dehydrogenase kinase, an enzyme critical to amino acid metabolism in humans. Defects in BCKDK are linked with some forms of autism.

A single residue change appears to confer the disease phenotype.

Creating new methods is great for three reasons:

1. It allows old phenomena to be explored in new ways.
2. It provides other researchers with more options.
3. It permits creation of ludicrous acronyms.

A recent paper by Moreno et al. in Molecular Systems Biology is an example of cases 1 and 3. Perhaps it will someday be useful to researchers studying protein-protein interactions, but for now, SPLIFF looks like a good way to visualize interactions found by other assays (i.e., yeast two-hybrid).

But I'm getting ahead of myself. This is a method called SPLIFF. It's about lighting up cells. Somebody got a little carried away with that acronym.

SPLIFF is a Split-Ubiquitin method and indicates protein-protein interactions within cells though the ratio fluorescence produced by two auto-fluorescent reporter proteins. Assays for protein-protein interactions usually use a growth phenotype or a colorimetric reporter to indicate interactions, so SPLIFF may offer more clearly quantifiable, temporally-sensitive results. The authors also show how SPLIFF, like other fluorescence-based methods, can localize interactions within cells. The protein interactions are reportedly detected instantaneously.  Hopefully this method can weed out some of the false-positives in two-hybrid interaction screens.

Citation:
Moreno, D., Neller, J., Kestler, H., Kraus, J., Dünkler, A., & Johnsson, N. (2013). A fluorescent reporter for mapping cellular protein-protein interactions in time and space Molecular Systems Biology, 9 DOI: 10.1038/msb.2013.3

Here's an interesting example of emergent properties.

In short, it's essentially a topology-based approach to solving a classical optimization problem. Quite novel, really, though it's not clear if the solution offers a whole lot of benefit over brute force calculation in terms of saved computation time. The paper is here.

What struck me as particularly interesting was the authors' proposal to physically implement their "blob" model:

...it would be satisfying if this virtual material could be implemented and embodied in a real physical substrate with the desired physical (for example visco-elastic, free energy minimisation) properties.

What would this accomplish? I'm not saying that it wouldn't be a neat, cheap engineering project to build an elastic blob capable of solving traveling salesman problems, it just doesn't seem like any kind of a job for real-world models.

Jeff Jones, & Andrew Adamatzky (2013). Computation of the Travelling Salesman Problem by a Shrinking Blob arXiv arXiv: 1303.4969v2

Here's a paper dealing with a frequently worrisome issue:

Shrestha, P. M., Nevin, K. P., Shrestha, M., & Lovley, D. R. (2013). When Is a Microbial Culture “Pure”? Persistent Cryptic Contaminant Escapes Detection Even with Deep Genome Sequencing. mBio, 4(2). doi:10.1128/mBio.00591-12
Microbiologists, like most biologists, like to have neatly organized categories and taxonomies available at all times. We work with individual species, specific strains of those species, and often mutants of those strains. If we grow our chosen organisms and they don't behave as expected, we generally assume that contamination occurred somewhere along the line or that a new mutation transpired. The usual preventative measure is simply to ensure that cultures start from single cells of the desired strain.

Shrestha et al. seem to have had some issues with culture contamination. This group works with Geobacter sulfurreducens strain DL1 in studies of microbial fuel cells. As they were attempting to isolate G. sulfurreducens strains with mutations enabling them to be more electrically conductive, they managed to isolate a strain later named KN400. This strain was great at conducting current but clearly wasn't just a mutant of DL1: sequencing showed that the new strain likely differed by more than 27,000 SNP's, or far more mutations than any lab-based methods of selection were likely to create.

The authors sought to clarify the origins of G. sulfurreducens strain KN400. Next-gen sequencing showed that a sequence specific to KN400 could be found in the original DL1 cultures, though out of more than 10⁷ sequence copies, fewer than 300 were the KN400 sequence. The new strain wasn't a new mutant at all. Subsequent growth studies found that even repeated rounds of restreaking cells on solid medium still yielded colonies with minute but detectable levels of the cryptic KN400 strain. Shrestha et al. suggest that their intense electrical conductivity-based selective pressures may have been the only reason they were able to isolate KN400 at all.

It's still not clear how G. sulfurreducens KN400 lives alongside the DL1 strain so well. My guess is that KN400 is actually an instance of a separate but phylogenetically related chromosome copy acting as a mobile genetic element. Cultures of the DL1 strain consistently maintain KN400 cells at low levels, so some variety of fitness benefit must be conferred to the entire culture or we'd expect the minority cells to just get selected against. There's clearly something strange going on with this bacterial species but it may be an ongoing process.

It was my birthday this past weekend! Danielle made a lovely cake:

You got your chocolate in my peanut butter, intentionally.

and a lovely phage:

Not pictured: crocheted phage in lysogenic state.

It also seemed like an appropriate time for this:

A birthday cake is no place for a cow.

Gatsogiannis, C., Lang, A. E., Meusch, D., Pfaumann, V., Hofnagel, O., Benz, R., Aktories, K., et al. (2013). A syringe-like injection mechanism in Photorhabdus luminescens toxins. Nature, advance on. Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. doi:10.1038/nature11987

Didn't read this one yet, but the abstract contains the phrase "vuvuzela-shaped channel" so it should be interesting.

Glud, R. N., Wenzhöfer, F., Middelboe, M., Oguri, K., Turnewitsch, R., Canfield, D. E., & Kitazato, H. (2013). High rates of microbial carbon turnover in sediments in the deepest oceanic trench on Earth. Nature Geoscience, advance on. Nature Publishing Group. doi:10.1038/ngeo1773.

This is a small letter about Challenger Deep, the deepest point in Earth's oceans. Located within the southern extent of the Mariana Trench, this spot is nearly 11,000 meters deep. It's pretty chilly down there - about 2.5 degrees C - but more importantly it's under extremely high pressure. Wikipedia tells me it's more than 16,000 psi or about 1,099 times surface pressure. This doesn't stop bacterial growth: Glud et al. found that two sediment samples from this deep spot contained, on average, nearly 10⁷ prokaryotic cells per cubic centimeter. Shallower sites nearby were also dense with microbial life but not nearly as rich as the Challenger Deep samples. 

Glud et al. suggest that deep-sea trenches like the Mariana may serve to naturally funnel fresh sediment downward, providing essential nutrients for microbial growth at extreme depths. Further analysis of these deep-sea microbes could show how they've adapted to such specialized metabolic demands. 

I went to a lecture today by Tim Read from Emory University. He discussed comparative genomics, specifically of Bacillus and Chlamydiaceae species and strains. The history of the latter is rather interesting: researchers thought these bacteria were actually viruses until the 1960s (due to the advent of electron microscopy, of course) as they couldn't culture them on artificial media. It's a group full of pathogens with wide host ranges but small, highly conserved genomes so it would be useful to know more about.

This review served as a basis for some new additions to the Chlamydophila psittaci Wikipedia page.

I've been thinking lately about that internet-adage, "if you're not paying anything, you're not the customer, you're the product." It comes up whenever someone, frequently the user of some free or freemium service, complains about sudden changes in terms of use or status of their service. The Instagram fracas last year and the Google Reader shutdown this year serve as good examples. Being a customer or a product of either of the two service may be possible but I'm not convinced that the above adage accurately describes any one individual's relationship with a company.

Every consumer is a customer and a product. We all do the whole exchanging-money-for-goods-and-services thing regularly. We all serve as marketing for these products and services as well. It might be the obvious tag on a pair of jeans or it may be more like membership in a club of fellow consumers (i.e.,  diehard Apple fans or anyone eating at Chik-fil-A after its recent PR issues). This marketing is provided free of charge. So are word-of-mouth recommendations, Facebook likes, discarded packaging, stock purchases, and any other interaction with a corporate entity, public or private. Even if all publicity isn't really good publicity, any continually increasing level of interaction with a company builds identity. That's almost priceless.

It's all just bacterial media. Photo from Flickr user Jepster.

Here is a paper:


Levkovich, T., Poutahidis, T., Smillie, C., Varian, B. J., Ibrahim, Y. M., Lakritz, J. R., Alm, E. J., et al. (2013). Probiotic bacteria induce a “glow of health”. (G. P. Kobinger, Ed.)PloS one, 8(1), e53867. Public Library of Science. doi:10.1371/journal.pone.0053867

This one looked like it was going to be silly. Most papers with quotations in the title tend to have that sense of "summarizing an idea with an adage" or just "excessive use of punctuation." Describing any observable phenomenon a "glow" rather than a glow really ought to give readers pause. We do live in an age when experimental animals can be engineered to literally glow. Is it a stretch to assume that the authors of this paper are trying out some new GFP-fusions with proteins indicative of healthy animal phenotypes?

Yes. This assumption would be wrong. This paper is about yogurt.

The authors claim that eating probiotic yogurt is associated with what they repeatedly describe as a 'glow of health,' sometimes with quotes and at least once without. I'll give them the benefit of the doubt as far as describing observable phenomena goes: the authors are all affiliated with MIT rather than Yoplait and they primarily discuss mice. For mice, the 'glow of health' includes thick, shiny hair and lustrous skin. These experimental animals were fed Lactobacillus reuteri within yogurt and on its own. As compared with control mice, the bacteria-eating mice were indeed significantly shinier and had thicker skin. All of these effects may be related to levels of the anti-inflammatory cytokine IL-10, as those 'glow'-ing traits weren't seen in mice without IL-10.


Some select quotes:

Taken together with our earlier data, this led us to postulate that probiotic bacteria induce host physiological changes including a more acidic pH resulting in radiant skin and shiny hair signaling peak health and fertility and thus a good reproductive investment.

(A good reproductive investment for the bacteria or the host?)

Extrapolation from data of mice to humans suggests that excessive inflammation in the form of uncontrolled IL-17A subverts scalp hair growth, and this may be remedied by eating probiotic bacteria such as L. reuteri, but interpretation is complicated by disparities in hair on scalp versus other body sites of these species [37]. Nonetheless, aged male mice eating probiotics displayed Il-10-dependent dense fur together with elevated testosterone and increased virility (data not shown) when compared with mice eating control diet alone. It is unknown whether eating of probiotic yogurt may forestall or reverse follicular activities of hormones in human subjects.  

This study leaves me with a bad taste in my mouth. The authors fail to address the actual differences between a probiotic-yogurt-based diet and the control diet; bacteria may just be more nutritive than the usual rat food. The sample size also doesn't appear to be larger than 20 animals in any one group. Last year's GM corn study had some similar issues. Even so, it's always interesting to see product claims (or even conventional wisdom, though I suspect it's still mostly marketing) put to the test.

I suppose the most important thing is just to know where to look.

The refrigerator room down the hall from my lab looks like it finally got fixed; it's constantly running at around 4 degrees C. Here's hoping it stays that way as it will make an ideal home for all our media.

I wonder how those rooms get installed in buildings.

The paper for today is:

Parks, J.M. et al. (2013). The Genetic Basis for Bacterial Mercury Methylation. Science, 339(6125), pp. 1332-1335. doi:10.1126/science.1230667.

(There is a tiny dog in this room and its constant yelpings and mewlings are making it quite hard to absorb this material.)

This is a quick little report about the genes used by mercury-methylating microbes to do their mercury-methylating work. The authors already knew about the corrinoid iron-sulfur protein (CFeSP) used to methylate metallic clusters on acetyl-CoA synthases, so they began to look for something similar in the genomes of mercury-methylating bacteria. They did find one within a Desulfovibrio desulfuricans genome along with a gene for a putatively associated ferredoxin. It's not just D. desulfuricans, either: the same pair of genes appears to be present in 51 other genome sequences, at least a few of which are known to be mercury-methylators. Beyond the bioinformatics, D. desulfuricans deletion mutants without these two genes appeared to grow normally yet had almost none of their usual Hg-methylating ability.

It is entirely possible that the two target genes in this study - referred to the authors as hgcA and hgcB - have some other functions. The genomes of some distantly related species do appear to contain hgcAB orthologs, including a set in the recently isolated Methanomassiliicoccus luminyensis, a commensal human gut-dwelling archaeon. It isn't clear why many of these species code for these enzymes or need anything like them at all. Studies of microbial metal metabolism look like another job for the "sequence everything that moves" approach.

Another Cytoscape quirk - when importing data, redundant pairs of interactions are usually - but not always - ignored. This is a problem when I actually want to see how many different sets of data produced the same interactions, so doing such a comparison may be a better job for Excel.

Cytoscape also has trouble importing VizMapper settings almost all the time. Sometimes importing the settings then saving and reloading the network irons out any bugs.

Edit: I figured out the problem with failure to recognize redundancy. Cytoscape ignores redundant entries if the interacting nodes are presented in the same order, so Node1 (pp) Node2 only gets added to the network once even if the data set includes it twice with different attributes, i.e. if Node1 (pp) Node2 from Data Set 1 and Node1 (pp) Node2 from Data Set 2 are both in the input file. If Node1 (pp) Node2 and Node2 (pp) Node1 are both in the input file, though, both interactions are retained despite just being the inverse of each other. A solution: import the attribute as an interaction type.

Argh, why is Google discontinuing Reader? Do people not use RSS feeds anymore? I know that the Big G really just wants to roll everything up into Google Plus Sign but they're hardly transparent about why they discontinue services like that. It's poor service (though not customer service, as evidenced by that internet adage along the lines of "if you're not paying anything, you're not the customer, you're the product).

An interesting note from a 2010 review of pathogenic E. coli by Croxen and Finlay in Nature Reviews Micro:

Intriguingly, EHEC can sense the hormones adrenaline and noradrenaline from host cells, as well as the quorum-sensing molecule auto-inducer 3 (AI-3) from gastrointestinal cells, to regulate motility and T3SS expression... Sensing of these molecules is required for virulence of EHEC in animal models and presents a new interaction that should be taken into account when considering pathogen–host interactions.

Some kind of a localization factor, perhaps, as adrenaline receptors are indeed present in the gastrointestinal tract.

It would be nice to actually accomplish something new in lab today but I'm spending an awful lot of time on small jobs like sending plasmid samples to labs in Austria and cleaning off plastic dishes so they can be sterilized (that job wouldn't even be necessary if said dishes didn't cost nearly $200 for a case of 60, but I guess lab materials are just obscenely expensive). There's also a bacterial pathogenesis exam tomorrow morning to study for. That material is generally fun to read, at least.

One hand washes the other.

I posted this generative text project on Facebook but it's something I'll want to come back to again. These kinds of project are just so neat in every sense of the word. I know they aren't that complex in their implementation but it's all a matter of presentation and how context deeply influences our understanding of even perfectly uninspiring text. The source is all here on Github. A selection (anything this project generates is a selection, anyway, as with the Twitter implementation it's nearly infinite, though the possible maximum number of tweets comes to mind):

...I saw stretched upon his back, and gazing up straight at the terrible sun, the man I was seeking. I saw about Miss Vivian's death to-day, and I was afraid Hal would be all alone fretting. I saw that William Oke, in his heart, thoroughly looked down upon all his neighbours. I saw only a snapshot of her, which showed her to be beautiful. I saw smoke in the chimney this evening. I saw a man talking with a woman there, at your door. I saw some beauties there a while ago. I saw tears in their eyes, tears of joy for the honors paid me; and especially, said they, for the manner in which I had received them. I saw her in the prisoners' dock, the Katusha betrayed by me, in a prisoner's cloak, condemned to penal servitude through a strange mistake, and my own fault. I saw them! The damage amounted to seventeen rupees, eight annas. I saw my Indian fall to the ground. I saw it in his desperate face. I saw my daughter at the theatre in London. I saw her only to love her; nor was it a common passion she inspired in me. I saw absolutely nothing else on the floor...

And so on. The Twitter version obviously doesn't benefit from being studded with excess punctuation but it does feel more like an authentic stream of consciousness, or maybe a stream of collective unconsciousness.

I went over to the MCV hospital cafeteria for lunch today. Lunch is quite inexpensive there due to the VCU discount, though the minor health risks of eating in a hospital are sometimes worrisome. Hospitals are just chock full of pathogens.

I saw Kristin and Joana from the Christie lab briefly while waiting in line at the cafeteria. Kristin said she referred someone to my lab who was looking for places for new microbiology graduate students to do rotations. It would be helpful to have some more help in the lab to handle small projects or even just to provide some fresh ideas. Hopefully any new students won't have to undergo extensive lab-skills training, especially if they're not going to stay around longer than the terms of a single rotation period.

At least I'm not the only victim of contamination right now:

Sergei Bulat,  a researcher at the Laboratory of Eukaryote Genetics at the St. Petersburg Nuclear Physics Institute, originally told RIA that they that "call it unidentified and 'unclassified' life."they found new signs of life after examining water samples from the subglacial Lake Vostok.

However, on Saturday the Eukaryote Genetics Laboratory head Vladimir Korolyov, spoke to Interfax News Agency, and said that they actually did not find any new signs of life. He said they just found contaminants.

Ugh, I got into lab today and discovered the following:

1. My yeast transformations performed last Wednesday appear to have yielded no transformant colonies at all.
2. The majority of the transformants for our last set of transformations may in fact be contaminants. 
3. My lab PI may not be in the office this week.

I'm becoming quite irritated with the research I'm trying to accomplish in this lab. These should be incredibly basic protocols but we're having little to no success at all with them. I'm almost completely out of variables to test and few of the options I've tested before have expanded our understanding of what is preventing our yest from transforming. All I know is that some of the plasmid stocks we already have are, in fact, incorrectly labeled. All this together is just impeding any process I could be making on an actual project.

Danielle and I were up in Delaware and PA this past weekend setting up wedding things. We got a site scheduled and a photographer booked, plus Danielle got measured for her dress. We also got to sit down and have brunch with the bulk of the Wedding Party Participants and sort out the basic details, i.e. when the event is and what they should (or shouldn't, mostly) be wearing at the time. We also went to see a movie with Danielle's mother and sister, which was watchable, mostly. Entertaining, perhaps.

(The movie was Oz: The Great and Powerful, which I will review parenthetically as follows: bright and colorful but predictable in Titanic proportions.)

This lab at UCLA is doing some pretty neat stuff with very small microscopes. I read about them in the Feb 2013 National Geographic but the article was very vague on details. They're working on developing mobile devices that can diagnose diseases like malaria using microscopy. Very useful.

I'm in Delaware for the day and I am currently petting a cat with one eye.

Today's paper is: 

Cissé, O. H., Pagni, M., & Hauser, P. M. (2012). De novo assembly of the Pneumocystis jirovecii genome from a single bronchoalveolar lavage fluid specimen from a patient. mBio, 4(1), e00428-12. American Society for Microbiology. doi:10.1128/mBio.00428-12.

Pneumocystis jirovecii is a fungus that isn't usually pathogenic but is deadly to immunocompromised patients, most prominently those with AIDS.  As with many other parasitic microbes colonizing human tissues, P. jirovecii can't be cultured in the lab. This disadvantage makes working with P. jirovecii nearly impossible if researchers seek to use genetic or molecular techniques. This looks like a case for sequencing! Wait, no, hold on. Researchers generally need to culture microbes to get enough genomic DNA for sequencing. The authors of this paper bypass that issue by collecting the fungus direct from the lungs of infected patients (they also used immunoprecipitation and random amplification, so this by no means an instance of single-cell sequencing).

Collecting samples of bronchoalveolar lavage fluid (or, if you prefer, the ridiculous-sounding acronym BALFs) provides plenty of fungus to work with. The samples are also rich in other genetic material from human cells, bacteria, viruses, and on occasion other species of Pneumocystis. The authors had to extract the P. jirovecii genome from the mess -- luckily, the genome of related species and rat pathogen P. carinii has already been sequenced and partially assembled. Process of elimination allowed for removal of the non-Pneumocystis sequences, then genome comparison and RNAseq helped finish and annotate the rest. More than just a de novo genome assembly using a messy metagenomic data source, this work is an example of how availability of massive quantities of genomic data make some projects entirely feasible.

It's a snowy, slushy mess out there! It's certainly been more wintry in Richmond before but rarely the kind of weather in which trees actively pelt me with slushballs on the walk to campus. It's still snowing, too. Danielle and I were planning on driving to Delaware this evening or tomorrow morning so I hope most of it gets cleaned up by then.

Today's paper is: Rao and Black - Structure and assembly of bacteriophage T4 head (2010) Virology Journal.

The T4 head structure. Looks kinda like a pineapple.

This is a review of the essential observations of the phage T4 head structure. Next to the famous lambda phage, T4 and its fellow T-even phages may be some of the most closely-studied viruses. T4 has that classic phage structure: an icosohedral head, a contractile tail, and a baseplate at the end of the tail, plus six spindly fibers on the baseplate. Cryo-EM has helped to define the structures of the primary T4 head proteins (gp23 and gp24) down to about 0.3 nm or 3 angstrom, which is a resolution that's hard to improve on. The gp23 protein demonstrates the classical HK97 fold and relies upon the E. coli chaperone GroEL and a phage-encoded cochaperone gp31 to reach its final form.

The paper digresses a bit to discuss phage display. I wasn't aware of how this technique was being used in recent years, but antigens fused to T4 capsids have been shown to be effective vaccines against foot and mouth disease virus, anthrax toxin and potentially even tumor metastasis. The authors aren't clear about how phage help with vaccine delivery. I suspect that they may have improved long-term storage capabilities when compared to antigen alone but the overall number of fusion proteins is limited by copy number per phage capsid; it can't really exceed the 870 copies of Soc already bound to the capsid and may also be limited by steric hindrance. Displaying antigens on phage rather than on their own may increase the immune response but I'm curious if it makes sense to use phage instead of a chemical adjuvant. The folks who did the anti-tumor study cite some references claiming as much and also claim that phage have the benefit of causing few side effects. I'd still call potential disruption of enteric bacteria a notable side effect.

Other interesting notes about the T4 capsid:

• The T4 packaging motor packages DNA at about 2 kb/s. It's an ATP-dependent process - no silly physics tricks here, though it's incredibly efficient (the authors state that this motor is "...approximately twice as powerful as a typical automobile engine", whatever they mean by 'typical'). The T4 genome is 171 kb so the motor gets the whole thing and then some packaged in less than 5 minutes. The authors must have summarized some numbers as a constant rate of 2000 kb/s should get it all packaged in less than 2 minutes, so the rest of the time may proceed more slowly or may be subject to something like strand slippage.
• The structure of the T4 portal complex is conserved across many phages and in HSV, which is really just an overgrown phage anyway. Studies really don't agree about how it works and I'm not inclined to bet on any one model at the moment.

A few departmental mailing lists just sent out a message about the Life Sciences building losing power due to a generator test between 1 and 3 PM. I really wish we were given more advance notice about these kinds of things.

Ugh, I'm fighting off a cold today. It's nothing serious, though sometimes I would rather have a decent excuse to stay home and avoid spreading disease, as I would with a slightly more virulent infection. It doesn't really help that I've been feeling directionless in the lab lately. I think one of the plasmid stocks is mislabeled and I can't seem to get our yeast strains to mate with any decent efficiency.

Today I discovered that a plasmid I thought was one thing was, in fact, another. I suspect it was mislabeled. I also suspect that I will take the blame for not verifying its identity sooner. At least we know now.

Danielle and I visited a couple yard sales and an estate sale this morning in search of a nice desk to replace the rather unstable Ikea one she is currently using. No luck, though it was interesting to see how many strange statuettes a person can accumulate in a lifetime.

In Cytoscape, you can select a subset of nodes including all edge types between those nodes by doing the following: use a filter to select nodes by edge type, then select all nodes connected by selected edges (or Ctrl+7). Follow by selecting adjacent edges (Alt+E). This will select a subnetwork determined by the filtered edge type but including the other edges (i.e. if you'd like to determine which nodes are connected by more than one edge of differing types). Don't forget to actually create the new subnetwork using the button on the toolbar.

Today I learned that Astrid doesn't handle shared tasks very well, despite this feature being central to its marketing. As with all free web services I can't complain too much. The problems aren't as apparent when using the iOS app and may in fact be solved by accessing the account through the app. It's hard to tell.

Hey blog. How are you doin'?

Today I was glad to hear that A Bit of Fry and Laurie was back on Netflix.