2016 is an important year for Nature Protocols as in the summer we will be celebrating our 10 year anniversary. Both in response to the success of some of our earlier protocols and reflecting the enormous improvements in the methods available to researchers over the past ten years, this month we are launching a new article type, the Protocol Extension.

Our first Protocol Extension is, as the name implies, still in the traditional protocol format. The method described in this article is based on an earlier version of the technique, for which we published a protocol back in 2006 from a different research group. This protocol is widely used by researchers and various changes to methodology have been made over recent years. Several of these adaptations are described in the new Protocol Extension article. Unlike Protocol Update articles, which should be used in place of the protocol they update, Protocol Extension articles provide a substantial modification or additional applications. They complement, rather than replace the earlier protocol, and may be authored by the same, or a different group of researchers. However, they also have some similarities to Protocol Updates, in that the Protocol Extension articles have headers to make their article type clear, they are always at the start of an issue, and there is a statement in the abstract to make it clear the article modifies or offers additional applications to one or more existing protocols.

As method development is an iterative process it is not always clear what constitutes an extension to a protocol. Very few new methods are developed that do not build on an earlier method, thus most of our protocols could be viewed as extending existing methods to a certain extent. In view of this we have decided to reserve this new article type for those articles where a new protocol is clearly warranted, and there is a clear reason to link the new article to an earlier protocol, for example, where sections of the procedure are the same.

As always, if you have any comments and suggestions, we would very much like to hear your views.

This month saw the announcement of the EMBO Young Investigators for 2014, and we’re very pleased to count three of them amongst our Nature Protocols Authors! All in all, 27 researchers from 11 different countries were selected to receive the status of EMBO Young Investigator this year; you can see the full list of researchers along with their research interests here.

So, we’d like to congratulate all the new EMBO Young Investigators, and especially our Authors Filippo Del Bene, Ines Thiele and Johannes Zuber. Ines has written two Nature Protocols on the topic of metabolism, one for generating genome-scale metabolic reconstructions and a second for predicting cellular metabolism using the COBRA toolbox, both of which have been very popular additions to our content. With a focus on RNAi, Johannes’ protocol provides a pipeline for the generation of shRNA transgenic mice, and Filippo’s protocol for converting zebrafish transgenic lines from eGFP reporters to GAL4 drivers with CRISPR/Cas9 is one of our most recent articles, published in Nature Protocols earlier this month. It’s great that we’ve been able to help disseminate the work of such up-and-coming researchers, and we’re equally pleased that they have found us the place to publish their protocols!

We are a little late to the party (it has been a busy month at Protocols HQ!), but Nature Protocols would like to extend their warmest congratulations to all this year’s winners of the Nobel Prize for Chemistry: Eric Betzig, Stefan Hell and William E. Moerner for their contributions to the development of super-resolution microscopy.

We are very pleased to have published a Nature Protocol from the Betzig lab earlier this year on Bessel beam plane illumination microscopy.  And we are equally pleased that the Protocol Exchange can claim a Nobel Laureate amongst its authors; the Moerner lab has published a guide to using Easy-DHPSF to measure the precise localisations of molecules in images acquired using a wide-field DH epifluorescence microscope. I would also encourage you to visit Moerner’s very informative lab website, if only to find out about the guacamole!

In the last month we have pushed live 9 new protocols on the Protocol Exchange!

– Long-term calcium imaging of ASJ sensory neuron controlling cold tolerance in Caenorhabditis elegans

– A spinnable and automatable StageTip for high throughput peptide desalting and proteomics

– Cold tolerance assay for studying cultivation-temperature-dependent cold habituation in C. elegans

– Protocol for delivery of macromolecules using dfTAT into live cells

– Mouse meninges isolation for FACS

– Microglial Sholl Analysis

– Modified paired end rapid library preparation protocol for 454 GS Junior 8 kb library preparation using Covaris g-tubes and BluePippin electrophoresis

– Purification of influenza virions by haemadsorption and ultracentrifugation

– Multi-parameter assessment of thrombus formation on microspotted arrays of thrombogenic surfaces

One of the limitations of the Protocol Exchange has been that it is difficult to work out how many times one of the protocols has been cited. We have recently realized that google scholar can capture this information, and it may be possible to extract a citation report by using the DOI as the search term.

For example:
If you wanted to find out how many times the protocol “Anisotropic Mobilities in Organic Semiconductors” had been cited, you could paste 10.1038/protex.2013.070 into the search field of google scholar.

The search will then give the following output:

I am not sure how this works and perhaps the data should be taken with a pinch of salt, but if you have an Exchange Protocol, you might want to perform the search and see what it spits out!

Congratulations to Nature Protocols authors James E. Rothman and Thomas C. Südhof, who alongside Randy W. Schekman have been awarded this year’s Nobel Prize in Physiology or Medicine “for their discoveries of machinery regulating vesicle traffic, a major transport system in our cells”, according to yesterday’s press release by The Nobel Assembly at Karolinska Institutet.

This year’s award acknowledges the fundamental contribution that research by Randy W. Schekman, James E. Rothman and Thomas C. Südhof has provided to the elucidation of the system that precisely controls the vesicle-based transport and delivery of cellular cargo within cells and from the cells to the outside environment. This traffic control is essential for the correct functioning of cells, and defects to it are known to occur, for instance, in diabetes and in a number of neurological and immunological disorders.

In detail, Schekman’s work has been instrumental in identifying the three classes of genes that control the cell’s transport system; Rothman, the author of two Nature Protocols articles, shed light on the mechanism by which the formation of specific protein complexes allows vesicles to dock and fuse with the correct target membranes; and, through his work of nerve cells, Südhof, the author of one Nature Protocols article, enabled the identification of the calcium ion–regulated molecular machinery that directs proteins to bind vesicles to the outer membrane of nerve cells, as a preliminary step to the release of neurotransmitters.

Dr. Südhof’s contribution to Nature Protocols is not specific to the research just mentioned, but it does provide detailed instructions on how to perform high-throughput gene expression profiling in individual neuronal cells using quantitative PCR. The approach covered by this protocol enables the investigation of hundreds of transcripts from a single neuronal cell, and it can be used to characterize, for instance, lineage-specific, reprogrammed neuronal cells.

One of the main areas of Dr. Rothman’s lab research is the protein superfamily known as SNAREs, which are present in yeast and mammalian cells. The key job of these proteins is to mediate fusion between vesicles and the cell membrane or the membrane of a cellular compartment. SNAREs are often subdivided into two categories, vesicle or v-SNAREs, which are embedded on the surface of vesicles, and target or t-SNARES, which are located on the surface of target membranes. Interaction between v-SNAREs and their t-SNARE counterparts directs the fusion of vesicles to target compartments through the formation of a three-protein complex formed by cognate SNAREs anchored on ‘opposing’ membranes.

The structure of a v-SNARE nanodisc. The green ribbons represent copies of a v-SNARE.

Dr Rothman has co-authored two protocols published in our journal, both useful for the study of SNARE proteins. A 2012 paper describes the preparation of fluorescently labeled v-SNARE liposomes and t-SNARE–reconstituted planar, supported bilayers that can be used to monitor docking and fusion events by conventional far-field epifluorescence or total internal reflection fluorescence microscopy. One year later, almost to the day, we published a second protocol co-authored by Dr. Rothman. In this article the authors detail the preparation of nanodiscs of fixed (small) size that contain fluorescent lipids and copies of a v-SNARE. Upon fusion of one such nanodisc with a liposome containing the cognate t-SNAREs, the occurrence of a fusion event can be quantitatively monitored following the increase in fluorescence caused by the dispersal of the initially quenched fluorescent lipids from the nanodisc into the non-fluorescent, and much larger, interior of the liposome.

The fusion process between a v-SNARE nanodisc (bottom) and a t-SNARE liposome (top)

Well, our heartfelt congratulations to Randy Schekman, James Rothman and Thomas Südhof for their achievement. And please forgive my lack of modesty when I point out that it also feels good to have it confirmed yet again that Nature Protocols authors are among the very best in their fields.

When I first mentioned to some colleagues that I was thinking of writing this post in the journal blog, a few quizzical expressions surfaced on the faces of Nature Protocols’ editors. After all, the ethical and philosophical implications of the protocols we publish aren’t the usual remit of Nature Protocols. Yet, when I found out that a method for mitochondrial DNA ‘transplantation’ introduced by Oregon Health & Science University’s Shoukhrat Mitalipov and co-authors is now technically almost ready for the fertility clinic¹, my mind started to wander into all kinds of questions and considerations that I thought I’d like to share with my colleagues and the readers of this blog.

In 2010, Nature Protocols published “Chromosome transfer in mature oocytes”², by Tachibana, Sparman and Mitalipov, an article that details the transfer of chromosomes from the mature oocyte of a rhesus monkey to the enucleated egg from a different rhesus monkey, so that the resulting oocyte has the nuclear DNA of a female primate and the mitochondrial DNA of another. This protocol was based on the ground-breaking 2009 Nature paper “Mitochondrial gene replacement in primate offspring and embryonic stem cells”³, which also reported that healthy monkeys had been born as a result of the procedure. These were monkeys that displayed no detectable presence of mitochondrial DNA of their biological mothers, but only that of the oocyte cytoplasm donor.

Mitochondria are often — and possibly too simplistically — called the cell’s power plants, because it’s in these cytoplasmic organelles that most of the ATP, the organism’s energy currency, is produced. Mitochondria have their own DNA (two to ten copies per organelle), which, as opposed to nuclear DNA, is passed en bloc from mother to offspring, without any paternal contribution.

Because of their role in ATP synthesis, mitochondria are exposed to a high concentration of free oxygen radicals, which, in conjunction with a lack of histones and limited mitochondrial DNA repair mechanisms, possibly explains why mitochondrial DNA mutations occur at a tenfold-plus rate compared with nuclear DNA mutations³. In humans, serious and often fatal disorders caused by mitochondrial DNA mutations affect 1 in ~4,000 children. Although current treatments alleviate symptoms and slow disease progression, no cures are available for these mitochondrial diseases.

At least in principle, a method that enables the complete replacement of mitochondria in the egg or embryo from a woman with known mitochondrial DNA defects with mitochondria from a donor with no such defects could act as an effective, ‘pre-emptive’ treatment of diseases linked to mitochondrial dysfunction. Just recently, Nature published another paper by Mitalipov et al.¹, which reports how the approach described in the Nature Protocols 2010 paper² has been successfully implemented to produce normally fertilized human zygotes that contained mitochondrial DNA only from the donors of oocyte cytoplasm (and not from the nuclei donors). These zygotes were found capable of developing blastocysts and of producing embryonic stem cells, which suggests that, if implanted in the womb, they could develop into healthy babies.

Of course, a breakthrough of this magnitude gives hope that a clinical application may not be too far down the road. This optimism is further encouraged by the significant success in achieving similar results in terms of mitochondria replacement in human embryos⁴ — albeit via a different approach — by a research group based in Newcastle, UK, and led by Professor Doug Turnbull. Incidentally, Dr Turnbull and co-authors published a method for the transfer of nuclear genome as a promising approach for the prevention of transmission of human mitochondrial DNA disease⁵ in 2010 in the Protocol Exchange.

The optimism, however, is tempered by substantial legal obstacles to the clinical application of these approaches. For instance, UK law currently forbids the genetic modification of human embryos or human eggs for treatment purposes, which prevents clinical use of both the approach developed by the UK-based group and the approach developed by the US-based one. In the US, the NIH restricts funding for research that destroys human embryos, so Mitalipov’s research group had to conduct its research using money from private donors⁶.

These legal and procedural hurdles are not mere technicalities, and a number of fundamental ethical and philosophical questions have to be univocally answered before the medical community embarks on the clinical use of pre-natal mitochondrial DNA replacement. Mitochondrial DNA only encodes 37 genes, or about 0.2% of our entire genetic make up. But the question still stands: what will be the exact relationship between a child and the woman to whom that child’s mitochondrial DNA originally ‘belonged’? Surely children who have not received their mitochondrial DNA from their biological mothers will look like their parents, but arguably efficient mitochondrial activity is vastly more important for the biology of a human being than the color of his or her eyes or whether his or her hair is straight or curly.

Furthermore, replacing a mother’s mitochondrial DNA does not make a difference just to her children, but given that it is passed down, more or less intact, from generation to generation along a matrilineal route, mitochondrial replacement may have permanent effects on many generations to come, including any possible unforeseen adverse consequences of the procedure. In order to observe, probe and record any such potential long-term adverse effects, subsequent generations of people who owe their mitochondrial genetic makeup to mitochondrial replacement will most likely have to be enrolled, basically at birth, in long-term, follow-up medical studies for decades to come; but presumably, once the age of consent has been reached, these individuals will have the right to refuse participation in such studies, won’t they? And what about their personal lives? Given the medical implications and the health concerns for their offspring, are the people involved going to be expected to disclose their genetic origin to partners they may want to have children with? A case could easily be made that the latter question applies only to female descendents of the woman who underwent the initial mitochondrial replacement. And shouldn’t the inherently discriminatory nature of this uneven burden be an additional cause for profound and unsettling moral questioning? After all, men are essentially genetic dead-ends when it comes to their mitochondrial DNA.

These are questions that no sensible person would ever volunteer to answer unless they had to, but we find ourselves exactly at that point in time. An answer must be sought and given, as the suffering of many men and women, children and babies may be avoided and their early death averted by the implementation of procedures to replace mitochondrial DNA. The UK government has launched a national, public consultation on mitochondrial replacement, which is to run until Friday December 7^th 2012, and which will advise whether change in legislation is appropriate. At the very least, this consultation must serve to stress the fact that in democratic societies, a decision on whether to go ahead, and in which terms, with the clinical application of these techniques, ultimately rests with the will of its members.

I argue that the scientific community at large should feel a particular responsibility to contribute to the moral, ethical, and philosophical discussion that is taking place. Scientists, researchers and science experts in general are among the people who are best equipped to know, understand, and anticipate the wide range of implications and ramifications of applying a technique such as mitochondrial replacement to the treatment of mitochondrial diseases. Ultimately, the Nature Protocols blog is as good an informal venue as any to discuss these matters and to air one’s views on the trove of thorny questions forced open by the publication, among other articles, of protocols in Nature Protocols² and the Protocol Exchange⁵…

1.                  Tachibana, M. et al. Towards germline gene therapy of inherited mitochondrial diseases. Nature https://dx.doi.org/10.1038/nature11647 (2012).

2.                  Tachibana, M., Sparman, M. & Mitalipov, S. Chromosome transfer in mature oocytes. Nature Protocols 5, 1138–1147 (2010).

3.                  Tachibana, M. et al. Mitochondrial gene replacement in primate offspring and embryonic stem cells. Nature 461, 367–372 (2009).

4.                  Craven, L. et al. Pronuclear transfer in human embryos to prevent transmission of mitochondrial DNA disease. Nature 465, 82–85 (2010).

5.                  Craven, L., Tuppen, H., Taylor, R., Herbert, M. & Turnbull, D. Pronuclear transfer in abnormal human embryos. Protocol Exchange https://dx.doi.org/10.1038/nprot.2010.54 (2010).

6.                  Cyranoski, D. DNA-swap technology almost ready for fertility clinic. Nature https://dx.doi.org/10.1038/nature.2012.11651 (2012).