In silico versus in vitro

For the past five days at least I have felt bad for not updating my blog with some fact about CRISPR or the latest controversy concerning “cancer stem cells”. The reason for this involuntary hiatus is, of course, lab work. All the lab work. All the time. How should I put this; there are distinct benefits to working in silico: leaving the lab/office whenever is convenient, being able to continue work at home, and actually getting home on time for dinner.

Here I am, genuinely happy to be doing work in cell culture (isn’t the dark blue of that lab coat excellent?):

image3

And then running samples in pre-cast 20-well gels ready to do a Western blot after having treated melanoma cells with a plethora of small molecule inhibitors:

image1

But then disaster had to strike. It was all going too well. Due to the bubbles rising from the electrodes in the above picture I didn’t have a clear view of the second gel running behind it. What a disappointment:

image2The sad smiley face accurately represents my emotions concerning this gel. Well, at least I know what I’m doing tomorrow.

So I’ll end with a mini CRISPR update: Tsai et al. (2014) developed a new method last year to reduce non-specific cleavage of DNA during genome-editing. They require expression of RNA-guided FokI nucleases, which also cleave DNA, but are only active when dimeric. Each single FokI molecule is guided to its target by a guide RNA, but only when two guide RNAs each bring a FokI to the desired locus do the enzymes become active. This drastically reduces off-target effects because both the sequence and spacing has to be correct. The original CRISPR/Cas9 system has considerable off-target effects, as shown by Lin et al. (2014), for example.

Reference:

Lin Y, Cradick TJ, Brown MT, Deshmukh H, Ranjan P, Sarode N, Wile BM, Vertino PM, Stewart FJ, Bao G (2014) CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Research 42: 7473-7485

Tsai SQ, Wyvekens N, Khayter C, Foden JA, Thapar V, Reyon D, Goodwin MJ, Aryee MJ, Joung JK (2014) Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat Biotech 32: 569-576

Seminars with Sirs

In addition to normal lectures and lab/project work, the M.Sci. biochemistry course here at Cambridge also includes weekly seminars, which focus either on a set of landmark papers or on a particular methodology. The general idea of these seminars is probably “to encourage students to think, to learn, and to think about learning, so that they ultimately develop the skill—and courage—to train themselves” (Raman (2015)). Raman argues in the eLife article that understanding past research in its historical context, whose results and implications are now taken for granted, is a key step to being able to come up with interesting questions and the appropriate experimental approaches to tackle these. Maybe in a few years we’ll know whether reading and discussing these landmark papers actually had this desired effect.

Two weeks ago the seminar was entitled “Greatwall and the control of mitosis” and was held by this charming fellow:

hunt

He is none other than (Professor Sir) Tim Hunt, who won a Nobel prize for Physiology/Medicine in 2001 together with Paul Nurse and Lee Hartwell for their “discoveries of key regulators of the cell cycle”. I would wager that the foundation experiments leading to this prize are taught in all biology undergraduate courses and so the seminar was not actually about these, but rather on the follow-up experiments conducted by Tim Hunt and his lab. In particular, the seminar was about the intricacies of cell cycle regulation by proteins called phosphatases. Phosphatases are enzymes that remove phosphate groups from other proteins and thus catalyse the opposite reaction of protein kinases, which add phosphates to proteins, usually at the amino acids serine, threonine or tyrosine. Some of what we discussed was summarised by Mochida & Hunt (2012), but the exciting and interesting parts of the seminar actually consisted of listening to Tim Hunt explain which experiments he agreed with and why, and perhaps more entertainingly, which experiments he does not believe and why.

Then a week later the seminar was hosted by John Walker:

walker_postcard

(Professor Sir) John Walker – surprise, surprise – also won a Nobel prize (Chemistry, 1997), together with Paul Boyer for “their elucidation of the enzymatic mechanism underlying the synthesis of adenosine triphosphate (ATP)”. [Incidentally, John Walker seems quite proud of being a knight of the British Empire: I caught a glimpse of the inside of the case for his glasses, “Sir John Walker + telephone number”.]

In this seminar we also briefly recapped the basics of ATP production by mitochondria, but again this is something we covered in first and second year. However, we then discussed the landmark paper (Abrahams et al. (1994)) describing the structural features of the enzyme ATP synthase that catalyses the production of ATP from ADP and phosphate. Incidentally, that paper was dedicated to Max Perutz for his 80th birthday, since he was involved in discussing this research at the MRC Laboratory of Molecular Biology. Subsequently, we moved on to more current topics relating to ATP synthase, such as its possible involvement in the formation of the mitochondrial permeability transition pore (Giorgio et al. (2013)).

Interestingly, although Hunt and Walker are of course entirely different people, there were two striking similarities between them: firstly, both of them are still active researchers who clearly are still excited by science and their experiments. Secondly, both are embracing new techniques and technologies, which were not available when they started out as scientists. For example, Walker and his group use molecular dynamics simulations (quantum mechanics and computation) as well as cryo-electron microscopy to study ATP synthase. Hunt also uses computational modelling to gain more insights into the complex networks regulating progression through the cell cycle, and Paul Nurse, who used to be a “simple” geneticist, has now essentially become a systems biologist. Hard work, joy at doing science and being receptive to new technologies all seem to be hallmarks of good researchers – best to bear this in mind.

References:

Abrahams JP, Leslie AGW, Lutter R, Walker JE (1994) Structure at 2.8-angstrom resolution of F1-ATPase from bovine heart mitochondria. Nature 370: 621-628

Giorgio V, von Stockum S, Antoniel M, Fabbro A, Fogolari F, Forte M, Glick GD, Petronilli V, Zoratti M, Szabo I, Lippe G, Bernardi P (2013) Dimers of mitochondrial ATP synthase form the permeability transition pore. Proceedings of the National Academy of Sciences of the United States of America 110: 5887-5892

Mochida S, Hunt T (2012) Protein phosphatases and their regulation in the control of mitosis. Embo Reports 13: 197-203

Raman IM (2015) Teaching for the future, Vol. 4.; eLife 2015;4:e05846

A bit about bacteria

Bacterial antibiotic resistance is a growing problem. This is due to several factors including, but not limited to:

  • The overuse of antibiotics to treat infections that should not be treated with antibiotics (apparently, one third of Americans believe that antibiotics can cure the flu).
  • The relative disincentive for pharmaceutical companies to invest in expensive and long-winded antibiotic drug development compared to cancer drug development, for example, because the latter drugs are used to treat a chronic disease whereas antibiotics are only taken by patients for a short time.
  • The drastic decline in the discovery of natural antibacterials since the so-called “golden era” of antibiotic drug discovery between 1945 and 1960, illustrated in this figure (copied from Wright (2007)) – note the large gap between 1962 and 2000:

Screen Shot 2015-02-01 at 10.55.00

Things are not all that bad, however, despite the fact that the word “pharmageddon” has been used to describe the scenario in which we have no effective antibiotics left to treat even the simplest bacterial infections. Only a couple of weeks ago Ling et al. (2015), reporting in Nature, described a new type of antibiotic termed teixobactin. [Comment on this paper can be found here.] In essence, they used the same conceptual approach as scientists in the 1950s: they “mined” for antibacterial compounds already existing in nature. The reason this has not been successful since the “golden era” is because 99% of bacterial species found in soil, for example, cannot be cultured under standard lab conditions, and so the vast majority of potential new drugs has been missed. Ling et al. circumvented this problem by culturing bacteria “in situ“, that is in their natural environment and/or with the addition of growth hormones. They then tested various compounds they found against Staphylococcus aureus and Mycobacterium tuberculosis and recovered teixobactin, which inhibits bacterial cell wall synthesis – eukaryotic cells (which include human cells) do not have a cell wall and thus the drug target is only available in bacteria. Teixobactin is produced by the bacterium Eleftheria terrae, which was not known to produce antibiotics. Hopefully this paper will a) fuel further research of this kind to uncover additional new classes of antibiotics, and b) lead to the development of teixobactin as a drug used in the clinic.

Although less directly related to bacteria, a new CRISPR paper was published a couple of days ago, also in Nature (Konermann et al. (2015); comment here). It demonstrates a proof-of-principle that the CRISPR/Cas9 system can also be used for transcriptional activation on a genome-wide scale, rather than genome-editing and gene knockout creation. The paper comes from Feng Zhang’s lab at MIT and in several ways is a follow-up on their previous publication (Shalem et al. (2014)): in the earlier paper they use CRISPR to knockout genes in the A375 melanoma cell line to establish which genes might be implicated in resistance to the drug vemurafenib. In this year’s paper they tested which genes, when overexpressed, led to vemurafenib resistance. Reassuringly, they found genes that were already known to mediate resistance, such as the EGF receptor and several G-protein coupled receptors, but also implicate new genes, such as those coding for ITG receptors, in the resistance mechanism.

References:

Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki O, Zhang F (2015) Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517: 583-588

Ling LL, Schneider T, Peoples AJ, Spoering AL, Engels I, Conlon BP, Mueller A, Schaberle TF, Hughes DE, Epstein S, Jones M, Lazarides L, Steadman VA, Cohen DR, Felix CR, Fetterman KA, Millett WP, Nitti AG, Zullo AM, Chen C, Lewis K (2015) A new antibiotic kills pathogens without detectable resistance. Nature 517: 455-459

Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F (2014) Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Science 343: 84-87

Wright GD (2007) The antibiotic resistome: the nexus of chemical and genetic diversity. Nature Reviews Microbiology 5: 175-186