Synthetic reactions inside detergent micelles: Bruce Lipshutz at the #ACSSanFran

On Sunday afternoon, I went to a symposium in honour of Bruce Lipshutz, who won the Herbert C. Brown Award for Creative Research in Synthetic Methods. The line-up of dynamic chemists and one chemical engineer!) was impressive and his work stood out as being truly ground-breaking.

He started by asking the question: Why do we do synthesis “the way we do”, when Nature does her reaction in an aqueous medium, at around 37 C, and using only trace amounts of metals for catalysis?

Organic solvents are the main polluters from synthetic chemistry and the number of years until many metals are extracted to exhaustion is sobering. While chemists don’t use these reagents lightly – it is not like its possible to magically make a lipophilic compound water soluble! And metals like palladium enabled reactions that were previous impossible – there is a move now towards developing new synthetic methods that address these issues.

For example, the green chemistry challenge from the US Environment Protection Agency who had a stand in the Exhibitor’s hall.


To address the solubility issue, Prof. Lipshutz’s team have developed a nanomicellar technology where organic reactions occur inside micelles formed from designer surfactants. Here water is not a solvent, but a medium – the reagents are not in solution, they are in a more dynamic state and are highly concentrated in the micelles. From the image below (taken from the Lipshutz Team website), the list of reactions that they have worked on is staggering.

nanomicelle small

They have been working on using iron nanoparticles doped with Pd, Ni or Cu to perform catalytic reactions such as Suzuki-Miyaura cross couplings, Sonagashira coupling, and reduction of nitro-containing aromatics.

What is notable, is that they are getting really good yields with lower catalyst loadings than those used in traditional organic synthesis; and that they are able to do may of the reactions – in some cases quite unexpectedly – under ambient conditions.

Synthetic collagen, protein microarrays and lipid bilayers – Sunday morning, #ACSSanFran

The “National Fresenius Award Symposium” celebrates researchers that have made amazing early career advances; this year the award went to Neal K. Devaraj, and this session was assembled in his honour.

The session started with a talk by David Chenowerth on making synthetic collagen. Collagen is a peptide triple helix made of peptide chains of the general form: [Gxx]3. His group has been doing experiments replacing carbon atoms with nitrogen – e.g. using aza-glycine instead of glycine in one (or more) of the repeating triplets Proline-Hydroxyproline-Glycine – which has remarkable effects on the stability of the resulting triple helices.

After a fascinating talk from Dale Poulter about exploiting the versatility of farnesyl transferase for the preparation of protein microarrays, there were three talks about lipid bilayers.

Steven Boxer spoke about recent work on tethered lipid bilayers – using a really clever approach using lipid-DNA conjugates with complementary DNA strands – which has been applied to looking at membrane fusion of enveloped viruses.

For imaging organelles, you could label either the membrane proteins or the lipids. An advantage of labelling the lipids is that there are many more lipid molecules than proteins in the membrane, so if you choose a photostable fluorophore that blinks spontaneously it should be possible to image for longer time periods before you get photobleaching. Alanna Schepartz spoke about work using an environmentally sensitive (fluorescesces more at low pH) fluorophore (Sir-Tz) to image things like Golgi and ER using STED.  The Supplementary Movies are remarkable both for the resolution, and for the length of time of the imaging experiment.

The last talk was by Neal Devaraj who spoke about his work on in situ synthesis membrane synthesis. So what does this mean? He creates clickable phospholipid precursors, sets up the reaction and watches how new lipid vesicles form over time. To solve the problem of catalyst dilution over time, they developed a really neat system for continual synthesis of a catalytic membrane.

Image taken from: 10.1073/pnas.1506704112, Hardy et al. (2015) PNAS 112  pp 8187-8192

They have also done some exciting work towards synthetic re-modelling of lipid bilayers using a reversible covalent coupling. Remarkably it is possible to see the formation of microdomains!

Image taken from: doi: 10.1073/pnas.1605541113, Brea et al. (2016) PNAS 113 pp 8589-8594

Writing home from the American Chemical Society Meeting in San Francisco

Dear Mother,
I have some exciting news! I am in San Francisco attending the American Chemical Society Spring Meeting.

acs picture

I made the rather unusual decision to stay at a youth hostel this time: the Pacific Tradewinds. As it turns out, there are quite a few attendees staying here – in fact 4 of my 5 room-mates are going to be walking with me this morning to the Moscone Center!

There are many parallel sessions, and we all have the impossible task of choosing between excellent options. The program runs to 607 densely printed pages; so getting the conference app was a big help.

Yesterday I discovered that there is this booth – called Café-X – in a building near the Moscone, where you can have coffee made and delivered to you by robot; so you can guess what I will be doing every day this week!

robot - small

Yours etc.

Introducing a third article type – the Protocol Extension

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.

SWATH-MS at Nature Protocols and Scientific Data

The lab of Ruedi Aebersold recently published a Nature Protocol for generating peptide libraries for targeted analysis of SWATH MS data-independent acquisition mass spectrometry. This protocol complements a data descriptor (published in Scientific Data by the same authors) for a large-scale human assay library that can be used to support protein quantification by SWATH-MS.

In order to understand an organism’s biology and how it responds to environmental changes (e.g. disease or drug treatments), many researchers would like to be able to correctly identify and even quantify as many of the proteins in any given sample as possible. Mass spectrometry – in particular liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) – is the analytical technique most commonly used for deep and reliable exploration of the proteome.

In bottom-up proteomics, the cells of the sample are lysed, the proteins are digested into shorter peptides, the non-peptide material is removed, and the peptides separated by liquid chromatography. In LC-MS, the column separating the peptides is interfaced with a mass spectrometer and the peptides are analysed as they elute.

Sometimes the researcher will already know which proteins will be most interesting for their study; in these cases Selected Reaction Monitoring (SRM) is a very sensitive mass spectrometry approach to detecting and consistently quantifying the peptides associated with these proteins over many samples. The reactions monitored are the specific fragmentations associated with peptides-of-interest that occur in the mass spectrometer.

In many cases this information will not yet be fully available before the measurement or the number of potential protein-players in the given system might be very large. Data-independent acquisition of mass spectra for proteomics experiments is an alternative approach that extends the number of proteins that can be targeted in a sample from approximately one hundred (in SRM) to several thousand. It is made possible by the development of mass spectrometry instruments that are able to acquire high-resolutioin mass spectra at a very high sampling rate. The analysis method requires the researcher to perform initial experiments to put together a library of time and mass spectrometric coordinates corresponding to the peptides associated with each sample type. These spectra are then used as the basis for the development of highly specific assays to detect and quantify the respective peptide in subsequent samples.

We recently published a protocol for generating peptide libraries for a type of data independent mass spectrometry developed by Ruedi Aebersold and co-workers called SWATH-MS. The assay libraries are generated by collecting high-quality fragment ion spectra in data dependent acquisition mode, and processing these in a number of computational steps as shown in the workflow below.

Workflow for SWATH assay library generation


Scientific Data published a data descriptor for a generic large-scale human assay library to support protein quantification by SWATH-MS. This library was prepared using the procedure described in the Nature Protocol and consists of 1,164,312 transitions identifying 139,449 proteotypic peptides and 10,316 proteins; it was generated by combining the results from 331 measurements of fractions from different cell lines, tissue and affinity enriched protein samples. 

The data itself is deposited in the ProteomeXchange at the following locations:
and at SWATHAtlas

Stem cells in Singapore

At the start of last month I headed to Singapore for the ISSCR Regional Forum “Global Controls in Stem Cells”. I had a great time and found the meeting a big improvement on last year’s Florence meeting, due to the increase in networking opportunities.

There was a lot of epigenetics at the meeting – as you’d expect given the topic focus. Several people showed the classic Waddington depiction of the epigenetic landscape and explained the need to define where in the landscape the various cell types present during human development lie, plus the different varieties of artificially engineered stem cells. It was fascinating to hear about the various different approaches being taken to address this question.

I don’t have the space to discuss every talk, so here is a brief summary of the sessions and some of the talks.

The first keynote of the meeting was from Rudolf Jaenisch. He started by talking about naïve versus primed embryonic stem cells (ESCs) and whether there is a single human naïve state. He then moved on to talk about finding disease-relevant phenotypes, for example using patient-derived induced pluripotent stem cells (iPSCs). He particularly focused on the need for an appropriate control, he favours an isogenic control to control for genetic background.

The first session was focused on reprogramming, with various researchers talking about their attempts to improve reprogramming and to dissect reprogramming pathways. Jonathan Loh talked about the miniaturised reprogramming system using 384 well plates his group has developed to aid this process. Hongkui Deng finished the morning talks with a fantastic talk about reprogramming human fibroblasts to hepatocytes with drug metabolic function. Up until now reprogrammed hepatocytes have lacked this function, which is so important if hepatocytes derived in this way are going to be used for drug screening.

After lunch there was a session on chromatin dynamics. Bing Ren focused on the functional analysis of transcriptional enhancers and developing a new way to look at gene regulation from a 3D point of view. Sung Hee Baek talked about chromatin modifiers in epigenetic control of cancer and stem cells. Ken Zaret talked about the initiation of cell reprogramming, specifically his efforts to identify “Pioneer factors”, these bind to target genes that will turn on later. The final talk in this session was from Ralf Huss, about using stem cells as drug delivery tools in cancer therapy, due to their propensity to home in on the tumour.

The first session of day 2 was devoted to the developmental biology of pluripotency. Ron McKay, one of the conference organisers, spoke on the need to understand the functional identity of human genomes. Many early lineage cell types can be grown, and the human ESC are all quite different. This variation needs to be understood. Sara-Jane Dunn talked about her computational work modelling pluripotency, an alternative approach to investigating this biological phenomena.

The first session after lunch was on Telomere and RNA biology. V. Narry Kim spoke first. The main focus of her lab is miRNA regulation, but in this talk she talked about RNA-binding proteins in ESC. Next up was Didier Trono who talked about how transposable elements and their epigenetic control mechanisms are key regulators of transcriptional networks in PSCs. Yue Wan talked about the importance of RNA structure in determining RNA function, and the need to learn more about RNA structure. Toshio Suda talked about the aging of hematopoietic stem cells. Stem cells are long lived, and they do change as they age. He talked about the different characteristics of “elderly” stem cells.

The first session of Friday morning was focused to Functional Genomics. Huck Hui Ng gave the first talk, on his work investigating the systems biology of stem cells. Andras Nagy talked about his groups attempts to produce a stable pluripotent cell quite different from ESCs. Frank Buchholz talked about using RNA screens to find modulators and regulators of the pluripotent state.

After lunch there was a Cancer session, covering a wide variety of aspects. Richard Young started the session with a talk about the roles of transcriptional super-enhancers in cell identity and cancer. Zsuzsanna Izavak talked about her recent Nature paper on transcription driven by primate-specific endogenous retrovirus HERVH in naïve stem cells. The HERV driven network partially explains differences in mouse and human stem cells. The significant differences between mouse and human stem cells were mentioned by several of the speakers throughout the conference. Jesse Smith talked about gene transcription inhibitors as potential cancer therapeutics. The rationale being that cancer can be viewed as a disease of de-differentiation, i.e. dysregulated gene transcription.

Yukiko Gotoh talked about regulation of neural stem / progenitor cell fate in the embryonic and adult mouse brain. The basic premise of her work is that embryonic neural stem cells build a brain, whilst adult neural stem cells have a very different role, e.g. in learning and memory.

Janet Rossant gave the closing keynote lecture. She talked about cells in early embryo development and studies looking at when developmental potential is lost, comparing the mouse and human embryo. She also emphasised the differences between mouse and human early embryo development, and the importance of remembering this when studying stem cells.

I very much enjoyed my time in Singapore – it really is a beautiful city. I attended the night safari on the conference networking trip, which was great fun and a really unique experience. I even managed to secure a gift for my children left at home – a lego “Mr Stem Cell” so I can introduce them to the delights of stem cells at an early age!

Meeting report: 4th Single Molecule Localisation Microscopy Symposium

In the shadow of The Shard at the end of August, I joined about ~120 scientists at KCL for the 4th Single Molecule Localisation Microscopy Symposium. This 3-day meeting preceded the announcement in October that the Nobel Prize in Chemistry had been awarded to Eric Betzig, Stefan Hell and William E. Moerner for their contributions to the field of super-resolution (SR) microscopy (which includes SMLM approaches), but it was already very clear from the content and calibre of the programme that the field was having an important impact on several key areas of biological research.

The Symposium was superbly organized by Helge Ewers, Mike Heilemann, Aleksandra Radenovic, and Jean-Baptiste Sibarita; the only real disappointment being the cancellation of Stefan Hell’s keynote lecture due to illness. The 25 talks were divided into 5 broad topics: technical developments; biological systems; quantitative localization microscopy; labelling; and data analysis. It was particularly pleasing from my perspective that women researchers were well-represented, with at least 1 female speaker in all but 1 of the high quality sessions. Nature Protocols authors were also among the speakers1, 2. The full schedule of talks can be found here.

I found most of the talks to be surprisingly accessible and, as someone very interested in techniques and methods, there was a lot to hold my attention. Much effort is being expended on improving SMLM with respect to: the achievable resolution, especially axially; its live imaging capabilities; and quantitative applications. Progress in all these areas largely relies on a combination of new microscope setups, improved labels, and advanced data analysis methods. Biological applications discussed included nanoscale clustering of membrane proteins (particularly during synaptic and T cell signalling in mammalian cells), molecular assembly/organisation of protein complexes, and single particle tracking. The talks were complemented by ~60 posters, which covered additional applications such as imaging the cytoskeleton, chromatin structure, and plant tissues.

But it wasn’t all hard work – attendees were treated to an evening boat trip along the Thames, which provided the opportunity for less formal discussions over dinner, whilst enjoying the beautiful views on offer. I spent a very enjoyable evening in the company of a lovely group of PhD students and young post-docs discussing life the universe and everything – and trying not to feel my age!

At the close of my 3-day crash course in SMLM, I certainly felt much better-informed and very much inspired by the amazing work being done by this small but enthusiastic community. It is certainly an area of research I will continue to follow closely – perhaps a trip to Bordeaux is on the cards for next year!


1. Quantitative imaging of membrane lipid order in cells and organisms Dylan M Owen,    Carles Rentero, Astrid Magenau, Ahmed Abu-Siniyeh & Katharina Gaus Nature Protocols 7, 24–35 (2012) doi:10.1038/nprot.2011.419

2. Direct stochastic optical reconstruction microscopy with standard fluorescent probes Sebastian van de Linde, Anna Löschberger, Teresa Klein, Meike Heidbreder, Steve Wolter, Mike Heilemann & Markus Sauer Nature Protocols 6, 991–1009 (2011) doi:10.1038/nprot.2011.336




EMBO Young Investigators

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!

Decoding neural circuit structure and function workshop

The last couple of months have been busy for the Nature Protocols editors, with Mel and I both travelling a fair amount. The end of September saw me heading off to Istanbul, Turkey for the EMBO workshop on “Decoding neural circuit structure and function”. I had been thinking a little warmth at the end of September would be a welcome change, but ironically I think it was actually warmer in London! And we didn’t really need all the rain to encourage us to attend sessions, given the excellent line up.

I don’t have the space to discuss all the talks at great length, and it was also encouraging to see so much unpublished data being presented, which I must not share. But here are my highlights.

The first talk of the meeting was by Juergen Knoblich, and it was great to hear about his current projects, using Drosophila genetics to understand cell cycle exit and quiescence plus the contribution of changing the balance of asymmetric versus symmetric divisions in development of various disorders. It was also good to hear that his group are doing more work on the brain organoids, whose creation has already been explained in their group’s recent Nature Protocols article. I was intrigued that a member of the audience asked about the ethical implications of such organoids – it was a question I asked myself whilst editing our article.

I enjoyed learning about behavioural assays using the larval zebrafish from Florian Engert. It was amazing to see what such tiny organisms are capable of. Scott Waddell’s talk was focused to resolving how an animal decides to do the most appropriate thing at a particular moment, using Drosophila as his model system. This question came up as the main research question being addressed in several speaker’s talks.

Another fascinating talk was by Leslie Vosshall on “Sensory cues driving host-seeking behavior in the dengue vector mosquito Aedes aegypti”. Leslie’s groups’ research should help address whether there is any truth in the various old wives tales concerning whether particular factors increase a person’s attractiveness to mosquitos. I spoke to some other researchers about Leslie’s talk and they are confident Leslie will be able to improve the repertoire of molecular biology research tools available for the study of Aedes aegypti. So, it is worth keeping an eye on what Leslie is up to.

The final talk of the conference was by Gero Miesenböck. I very much enjoyed the pictures of the T-maze apparatus his group uses to perform mass behavioural assays in Drosophila.

In addition to all the fabulous talks there were stimulating poster sessions. I particularly liked the poster by Deniz Atasoy from Medipol University, Istanbul, on a Genetically encoded synaptic marker for electron microscopy (GESEM) that he developed. This poster was awarded the Nature Protocols poster prize, going to the poster with the best protocol behind it. Special mention should also go to Xuefan Gao from the Max Planck Institute for Developmental Biology in Tubingen, whose poster on AVEXIS, a protein-protein interaction screen assay, was also very methodological in slant.

Do let us know if you are organising a conference and would like to have a Nature Protocols poster prize as we are always looking out for opportunities to sponsor such prizes.

We also enjoyed conference dinners at Boğaziçi Üniversitesi and on a boat on the Bosphorus – so it wasn’t all work! The conference organisers, Arzu Celik, Nilay Yapici and Hernan Lopez-Schier, deserve a special thank you for being so welcoming and organising such a good program. Istanbul is a beautiful city and well worth a visit.

Modelling the molecules of life – a talk by Michael Levitt

Last thursday I went to an inspiring talk given by Michael Levitt, joint winner of the 2013 Nobel prize for chemistry. It was the 2014 Sir Ernst Chain Lecture at Imperial College, London.
Starting with a project using lysozyme (where the co-ordinates for the computer input came from a ball and stick model and were typed onto punch cards) and early computer simulations of protein folding, to his more recent work on modelling ribosomes and eukaryote chaperonins, he presented a small slice of the amazing work that he and his collaborators have done. What was equally evident was the phenomenal advances in technology and computational power over the last 40 years.
He is currently holding a chair in Cancer Research at Stanford, and spoke a little about the work on humanised antibodies that he did with Cary Queen  at Protein Design Labs (a company that formed in 1987, is now PDL biopharma  and produced patents that underpinned the development of drugs like Herceptin,and Avastin).

He included a slide providing advice to the young:
Be passionate
Be persistent
Be original
Be kind and good (or, at least, act kind and good…)

He thanked the Nobel Committee for Chemistry for, amongst other things, considering awarding a prize for a method rather than a solution; a sentiment especially pleasing to the editors of Nature Protocols!

Some of the slides were similar to those published here.
In 2003, he prepared a series of video talklets to form a web-based class on Computational Structural Biology. These are available on-line; and I plan to listen to as many of these as I can this coming weekend! Structural Biology 228 | Computational Structural Biology