Making the hemin stock/solving a chemical puzzle

Yesterday I tried, and failed, to make a new lab stock of the hemin that Haemophilus influenzae needs as an iron source. 

Hemin comes as a fine black powder; it's not really soluble in aqueous solutions or even miscible in water.   For our culture media it's prepared as a sterile suspension by mixing 100 mg of hemin with 100 ml of a solution of 4% triethanolamine (a surfactant), and incubating this at 65°C in a waterbath for 30 min.  The hemin forms a black suspension that's, rather surprisingly, both sterile and chemically stable.  Sterility is surprising because 30 min at 65°C isn't expected to kill spores, and stability is surprising because, once the hemin is added to culture media it goes off within 48 hr.

Anyway, we're down to our last 100 ml bottle of hemin stock, and it's my turn to make up lab stock solutions, so yesterday I tried to make more.  We've always used triethanolamine that came as a viscous liquid, but recently we accidentally purchased triethanolamine hydrochloride, which comes as a white powder.  So I made up a 4% solution (nicely soluble) and mixed it with the hemin.  NO SUSPENSION, even after heating.  No different than hemin in pure water.

What to do?  Was the problem the triethanolamine or the hemin, which was also a new bottle (well, a bottle of uncertain age that had never been opened).  The new and old hemins were both from Sigma and had identical labels (the old one in a brown glass bottle, the new one in a white plastic bottle), so that's probably not the problem.  Our ancient CRC handbook (the 'rubber bible') recently disappeared, so I looked triethanolamine up in the Merck Index.  Not much help, so I just left the problem for the next day, thinking I'd email the building to see if anyone had any liquid triethanolamine.

But this morning I've done some Google searching and discovered that liquid triethanolamine is mildly alkaline, but solutions of the hydrochloride are quite acidic.  Triethanolamine hydrochloride is used in various molecular biology and microscopy protocols, always with the pH adjusted to 8.0.  So this morning I'm going to try adjusting the pH of the 4% triethanolamine before I add it to the hemin.

Later:  And here's the result!  Raising the pH caused the hemin to form a nice evenly black suspension (bottle on left).  The bottle on right has its original low pH and all the hemin has settled to the bottom of the bottle.

Informing authors of the real consequences of CC-BY open-access publication

Thanks to comments on the previous post, I now realize that there have been extensive discussions of the merits of different CC licenses for open-access publishing.  See for example: http://oaspa.org/why-cc-by/. These discussion explain that the most open licenses are best for the dissemination and utilization of scientific information. That's probably why both PLOS One and BioMed Central use only the CC-BY license, which allows unlimited use and modification, including commercial uses, provided the source is attributed.  At a minimum the attribution requires listing the names of the authors.  It should also require citing the journal where the article appeared, although this isn't always clearly spelled out.

 

But, as the previous post and a related post describe, the CC-BY license creates new problems for authors, because some for-profit publishers have begun aggregating CC-BY papers into high-priced edited books without the authors' knowledge.  The authors I've discussed this with are quite upset.  They trusted the journals to offer licensing arrangements that were in the authors' best interests, but now they feel that they have relinquished control of their scientific reputations.  (Note that these weren't predatory publishers, but PLOS One and BioMed Central.)

Most of the discussions of open access licenses haven't considered the exploitation of these licenses by for-profit publishers, probably because this niche opened only very recently, once open-access papers became widely available.  I and others discovered this problem by accident.  I don't know how widespread it is, but I expect it will only grow.  (I'd like to do a survey of its prevalence, but I can't figure out any way to distinguish between such repackaged books and traditional multi-author volumes without having to contact individual authors - any suggestions?)

So far, open access publishing has been presented as both a public and a private good.  Science benefits from barrier-free dissemination and reuse, and authors benefit from wider readership. The only cost under discussion has been the transfer of publication expenses from the reader to the authors or their institution.  Opposition has come mainly from the publishers of subscription-access journals whose profits are threatened.

But for-profit republication of open-access articles is a cost that most advocates of open access didn't anticipate and that most authors are unaware of.  It's time for open-access advocates and especially publishers to take on the responsibility of informing authors of all the consequences, not just the good ones.

In some ways the ethical issues are like those of a clinical trial.   Participation generates a public benefit (medical research) and may be directly beneficial to the participant (better medical care, access to new therapies).  But the researchers directing a clinical trial are obliged to make sure that potential participants also understand the risks and costs.  They can't assume that the participants have thought the implications through, but must spell these out in clear and simple language.

Similarly, advocates of open access need to honestly inform authors about the consequences of the CC-BY license.  The onus shouldn't be on the authors to research the implications and consequences of different licenses, but on those with expert knowledge to communicate this to the authors. 

So, two questions:

Should authors in open-access journals be allowed to choose between different CC licenses?  Publishers agree that CC-BY is best for science, but authors may think it is not best for them. The major publishers don't give authors any choice, but I think they should.

How should open-access journals inform authors about license consequences?  This is particularly important when CC-BY is the only or default license.  Most scientists I've talked to are unaware that the CC-BY licenses of their open-access papers allow commercial publishers to alter and republish their papers without consulting them.  And they are very unhappy to learn that this is actually happening, often saying that they'll have to rethink their use of open-access.

Here's what the authors are told at present.  Note the emphasis on benefits and no discussion of risks:

PLOS One:
"Upon submitting an article, authors are asked to indicate their agreement to abide by an open access Creative Commons license (CC-BY). Under the terms of this license, authors retain ownership of the copyright of their articles. However, the license permits any user to download, print out, extract, reuse, archive, and distribute the article, so long as appropriate credit is given to the authors and the source of the work. The license ensures that the article will be available as widely as possible and that the article can be included in any scientific archive."

BioMed Central:

"You retain copyright, licensing the article under a Creative Commons license: This means that articles can be freely redistributed and reused as long as the article is correctly attributed. For example, a published article can be posted on a personal or institutional homepage, emailed to friends and colleagues, printed and sent to people, archived in a collection, distributed on CD-ROM, included in course-packs, quoted in the press, translated and further distributed as often and widely as possible. Read the full Creative Commons license."

When is it ethical to re-publish open-access scholarly articles?

Note added October 7, 2017:  Apple Academic Press has fully addressed the concerns raised below.  Their practices now ensure that authors are appropriately consulted and credited when their open-access articles are republished. 

A colleague just discovered that an article she published in the BioMed Central journal Epigenetics and Chromatin has been republished, without her knowledge or consent, in a book, Epigenetics, Environment and Genes, 'edited' by Sun Woo Kang and published by Apple Academic Press.  You can buy it on Amazon for $104.26 (Can).  

On checking the details of the BioMed Central publication agreement, we discovered that this commercial reuse is permitted.  It's also permitted by the PLOS One agreement, though that is CC-BY which I think requires that the original publisher be credited as well as the authors.

At first we thought that all the reprinted articles were from open-access journals, but at least one isn't.  I've contacted the author of that article to find out if permission was obtained for its republication.

Apple Academic Press sent this reply to an email from the authors:

I am answering the concerns over the reuse of your article in Epigenetic, Environment and Genes book published by AAP. 

This book was contracted through "Harding house publishing services" based out of Vestal, New York. The editor and contractor were responsible for the selection of articles in the book and to our knowledge this article was used under Creative commons license. By allowing your article to be printed by open access journal that lists the Creative Commons license, you as the author are agreeing to those conditions. The rationale for doing so would be to allow your work to be distributed in as many ways possible, which is common practice in the world of academic publishing.  http://creativecommons.org/licenses/by/2.0

However, that said, at Apple Academic Press we agree that authors should be notified whenever their work appears in a new publication. It is our current policy to contact all authors before including their work in any of our books. We apologize that this policy had not yet gone into effect when the edition of Epigenetic, Environment and Genes  was in production, but we have rectified that now. We would be happy to send you a copy of Epigenetic, Environment and Genes.
I have copied Ellyn Sanna (President and CEO) of Harding House publishing, So that she also gets aware of your concerns. I am sure if you have any more questions, she will be happy to take this further with you. 

Also, Our product line is growing rapidly, and we are looking to work with new authors in the STEM subjects. If you have any book concepts for your field (whether originally authored or compilations of recent research), we would be very interested in working with you in that capacity as well. Our authors and editors receive generous royalty contracts.

I particularly like their brazen invitation at the end and the offer of 'generous royalty contracts'.

My colleague and her co-authors are outraged; they had no idea that this commercial reuse was permitted by the standard agreements.  In principle the authors should have read the fine print, but as advocates of open-access publishing I think the onus is on us to make sure that the copyright agreements are in accord with authors' reasonable expectations and wishes.

At a minimum the open-access journals should inform their authors that unscrupulous publishers are standing ready to exploit their work.

We're not the first to note Apple Academic Press's sleazy work.  Read this post written by Mike Taylor a couple of weeks ago, complete with a nasty response from a former employee of the publisher.  And this post by Christopher Schadt from a few days before that, which has a long comment thread about who has the rights to do what.

My turn to do lab meeting

I've been so absorbed in my Useful Genetics course that I've let our weekly lab meetings lapse over the last few months.  But I've set up a new schedule and today it's my turn to present.  Of course I haven't done any experiments lately, but I've done one tiny analysis and I'll also talk about plans for the CIHR proposal.

The tiny analysis was the first step in addressing a question I've been wondering about for a while now- the phylogenetic distribution of the rec2/comEC gene.  In H. influenzae the  Rec2 protein sits in the inner membrane and translocates a single strand of DNA from the periplasm to the cytoplasm.  Its Bacillus subtilis homolog ComEC does the same thing at the one cytoplasmic membrane. 

Homologs of these proteins are present in all known competent species, and the appropt=riate experiments have been done they have the same DNA-translocation function, are competence-regulated, and do not have any other obvious function in the cell.  This distinguishes them from all the other proteins consistently required for DNA uptake, which are members of the type 4 pilus complex and known to also function in other pilus-associated processes.

Thus the phylogenetic distribution of rec2/comEC might be expected to reflect the phylogenetic distribution of competence.  But as far as I know nobody has examined this.

Before I describe what I found, I should bring up the distribution of another competence-induced gene, dprA.  I've written about dprA here and here; it doesn't contribute to DNA uptake but protects incoming DNA from nuclease degradation and promotes homologous recombination with the chromosome.  I previously discussed the phylogenetic distribution of dprA; it's very widespread and highly conserved across a wide range of bacteria not known to be competent, so I argued that it must have another function that's independent of promoting homologous recombination of incoming DNA in competent cells.  I was leaning to the idea that 'competence-regulons' include not only DNA uptake machinery but machinery to mitigate other harmful consequences of nucleotide scarcity, especially stalling of replication forks.

Back to rec2.  My tiny analysis was to take a first look at the distribution of rec2.  All I did was ask Gen Bank to search for rec2 and for comEC.  As controls I used dprA and recA.  GenBank helpfully provides a tree-view summary of what it finds, and that's all I've looked at.  Here are the results of the four searches:

 

First conclusion:  rec2/comEC homologs are present in many many bacterial genomes than there is any evidence of competence for, including some that have been shown to not take up DNA.  This could have one of two interpretations.  First, it could mean that many many more bacteria are naturally competent, and that competence is likely ancestral to all bacteria.  Or, it could mean that rec2/comEC has another function in the cell, one that's independent of the ability to take up DNA.  

Note the similarlty of the comEC and dprA distributions.  (I suspect that most of the rec2 hits are also included in the comEC results.)  I had only been thinking of the first interpretation for rec2/comEC, but had previously taken the second interpretation for dprA. But now I think I need to consider both interpretations for both genes.

Reclaiming my explainer energy (from MOOCing back to blogging)

My Useful Genetics MOOC (massive open online course) isn't really over yet, but almost all the work is done so I'm finally able to think about research.  And it's high time, because we have two research visitors in the lab for the next few months, the post-doc and I have two reviews to write, and I need to write a grant proposal for a Sept. 15 deadline.

I'd better start with the proposal.  We've had 5 (or is it 6?) unsuccessful tries to get CIHR funding for work on the mechanism of DNA uptake so, even though one of our visitors brings expertise that would help with this, I've given up hope of ever getting serious funding for this work.  We then made one try to get funding for a new project, aiming to predict recombination in natural populations.  This was solidly rejected.  So now I'm going to go back to our funded strength, the regulation of competence.
We have several papers in this area recently, and lots of directions to investigate.

Here I've pasted directions onto our standard diagram of competence regulation - all the blue boxes are regulatory problems in serious need of investigation.  I won't necessarily include them all in the proposal, and there are probably other problems I've forgotten about, but here are the basics:

1. Fructose:  Competence is regulated by cAMP levels which are elevated by the H. influenzae phosphotransferase system (PTS) in response to depletion of the sugars that the PTS would otherwise transport.  In H. influenzae the only such sugar is fructose.  Regulation by fructose levels in the host environment makes no regulatory sense.
2. Sxy-CRP interactions: We've done a lot of work on these, and by we I mean the former RA (she has a great new job in a high-powered lab across the street) and former grad student/postdoc (he has a great new job at the University of Regina).  But we still don't know what's going on.
3. Hfq:  Hfq is important for the activities of small regulatory RNAs. Knocking out Hfq reduces competence 10-fold, by an unknown mechanism that's independent of the purine-repression effect described below.
4. Toxin-antitoxin system: One pair of genes regulated by competence turns out to encode a probable toxin-antitoxin system, with the antitoxin preventing the toxin from preventing DNA uptake and transformation.  We have no idea  whether this futile combination has any biological function.
5. Regulation by purines: We have a nice new paper out about the purine regulation, showing that it acts by stabilizing the sxy mRNA secondary structure that blocks translation.  But we have only very indirect evidence of how it does this.
6. Effect of PRPP on sxy mRNA translation:  The indirect evidene suggests that purines may regulate sxy mRNA translatability by changing the intracellular level of the intermediate PRPP (phosphoribosyl pyrophosphate).  We probably can't investigate this biochemically, because PRPP is not something you can buy (probably very unstable?).  But maybe we can investigate its role genetically, by making more mutants.
7. Stalled replication forks:  We have been hypothesizing that one function of the competence regulon's proteins is to stabilize replication forks that have stalled because of a shortage of nucleotides.  One way to test this is to see if hydroxyurea induces either the regulon or competence, because hydroxyurea inhibits the enzyme ribonucleotide reductase, which is needed to convert NTPs to dNTPs for DNA synthesis.  I'm told that hydroxyurea is known to cause stalling of replication forks, though I haven't looked for this yet.
8. murE mutants: Point mutations in the cell wall biosynthesis gene murE cause constitutive expression of the competence regulon.  We have no idea why.