A few weeks ago, Jason Kelly explained in his post how Itaya and colleagues (2007) assembled the complete 135 kb rice chloroplast circular genome starting from a collection of 5-6 kb fragments and using sequential in vivo homologous recombination in Bacillus subtilis. Now, Hamilton Smith, Craig Venter and colleagues have achieved the assembly of a complete 583 kb Mycoplasma genitalium genome ("JCVI-1.0", Gibson et al, 2008). The starting fragments were of similar length, 4-5 kb fragments with 80-360 kb overlaps, albeit synthesized chemically rather than by PCR. In contrast to Itaya et al, Ham Smith's team used in vitro recombination (using T4 pol digestion/annealing/Taq pol repair and ligation) in a 3 step hierarchical assembly process and completed the fourth step, the assembly of 4 quarter genomes, using in vivo homologous recombination in yeast (TAR cloning, Larionov et al, 1996). The use of yeast for the last step might be a little worrying, given the high recombination activity in yeast and the propensity for large constructs to rearrange (I used to work with YACs to construct mouse transgenes and I can still feel the pain... but I don't know about the stability of circular TAR clones). In any case, it worked! One final clone was sequenced (7X coverage) and, remarkably, was shown to match exactly the sequence designed!

This impressive technical feat may eventually have tremendous consequences when combined with the transformation procedure ("genome transplantation", ) Venter and colleagues reported last year (Lartigue et al, 2007). As Dawkins noted at the Digital Life Design meeting in Munich a few days ago (see video below for some excerpts of his discussion with Craig Venter and the transcript in Edge), "genetics has become a branch of information technology".

JCVI-1.0 has obviously not been assembled "from scratch". In fact, beside some "watermark" sequences inserted to distinguish the synthetic genome from the native one, the fact that its sequence is a remarkably accurate copy of M. genitalium genome is probably one of the major achievements of the study. The technology for the synthesis of very long DNA of arbitrary sequence (in principle...) is thus progressing at an impressive pace. But writing a genome is not (yet) equivalent to designing it. Exciting (and hard) work remains to be done to bridge this gap and to improve our understanding of how biological functions can be created by assembling genes into a synthetic genome and developing the tools that will make this process rational and efficient, a challenge the synthetic biology community is eager to tackle (see The BioBricks Foundation)...

(via Scintilla)

The article on "Probiotics modulation of mammalian metabolism" published this week in Molecular Systems Biology by Jeremy Nicholson and colleagues (Martin at al, 2008) has attracted some attention (read the nice summary in Science News) in some (very) popular media (here, here, here and here).

In this follow-up study of the paper published last year (Martin et al, 2007), the team lead by Jeremy Nicholson, in collaboration with Nestlé, demonstrates clear physiological effects of oral probiotics administration on mice harbouring a humanized microbiome. The effects are intricate: both the host flora and metabolism are altered. By analyzing metabolite pools in several compartments (liver, blood, urine, feces, gut), and following in parallel the host microbiota, patterns of correlations between microbial species and metabolites start to be visible and reveal the probiotics-induced modulation of the microbial-mammalian interactions. But the actual paper is really just next door (synopsis), so have a look...

How will these results translate to humans? What will be the best way to influence our microbiome? Drugs or yoghurt? These are fascinating questions and the understanding of how our physiology depends on the microbial flora could have profound consequences, particularly in these times when we seem to be in a "rush to gene-based solutions to all our problems" (Wilson, 2007). Will personal genomics have to ultimately develop into personal metagenomics to include our "extended" microbial genome?

Even if I usually prefer to resist the temptation of a self-promoting section in this blog, I find the attention of the media for this topic interesting (despite the usual variable accuracy of newspaper reports) because it points to an area where systems biology provides insights into topics of immediate interest to the general public.

The NIH has recently started its Human Microbiome Project. In this context, this study also underscores the importance of developing model systems and tools to manipulate the microbiome and to analyze the incredibly dense and intricate interactions that connect host and microbial species. A field where top-down systems biology seems indeed a very pragmatic and promising approach.

A controversy seems to be brewing over some recent theories and quantitative analyses addressing the fundamental question of how the Bicoid morphogen gradient is established and decoded in early Drosophila embryos. The transcription factor Bicoid controls the anterior-posterior patterning of the developing embryo. It is translated from maternal mRNA localized at the anterior pole of the egg and its graded distribution activates, in a concentration-dependent manner, the expression of gap genes, thus determining their spatial domain of expression. Synthesis from a localized source combined with diffusion and uniform degradation of the Bicoid morphogen provides one of the simplest models to explain the approximately exponential shape of its gradient. While, historically, patterning has been thought to rely on the gradient at its steady state – that is when synthesis, transport and degradation processes balance each other – the question arose as to whether steady-state can be reached rapidly enough in the quickly developing embryo (Lander, 2007).

In February last year, Naama Barkai and colleagues published a study (Bergmann et al, 2007) in which they propose that the gradient would in fact be interpreted before it has reached its steady-state, when the gradient is still "moving". Experimental evidence for a dynamic evolution of Bcd profile between cleavage cycle 11 and 12 is provided using a reporter gene driven by bicoid-binding sites. These authors further show that a pre-steady-state model implies a reduced sensitivity of the gradient readout to variations in the production of morphogen at its source. One biologically relevant example of this robustness is the observation that the domain of expression of hunchback, a Bicoid target gene, shifts much less in embryos from mothers with altered bicoid gene dosage than would be predicted by a steady-state model.

A few months later, Thomas Gregor and colleagues published two papers (Gregor et al, 2007a, 2007b) reporting a detailed analysis of the profile and dynamics of the Bicoid gradient. Quantitative in vivo imaging of a transgenic bicoid-eGFP reporter revealed several paradoxes. While a stable gradient of nuclear Bicoid is quickly established (within 90min, approx. cleavage cycle 9), the (local) diffusion coefficient of Bicoid, as deduced from photobleaching experiments, appears to be far too small (D=0.3 μm²/s, much less than expected from previous estimations made by injecting labeled dextran molecules) to be compatible with such a rapid establishment of the (long-range) gradient by diffusion alone. These experiments further show that nuclear Bicoid is under a highly dynamic nuclocytoplasmic equilibrium, pointing to a fundamental role for the nucleus in gradient establishment and stability. Finally, the precision with which the Bicoid gradient is transformed into Hunchback expression (see illustration, after Gregor et al 2007b) is estimated to be around 10%. This remarkable level of precision would not only be close to the physical limits of the system, but also strikingly matches the accuracy required to detect changes of Bicoid expression between adjacent cells (10%, equivalent to a difference of only 70 Bicoid molecules per nucleus) and the level of reproducibility of the absolute morphogen concentration from embryo to embryo (10% as well).

In a Correspondence published last week, Bergmann and colleagues (2008) dispute these interpretations and claim that a "reanalysis of their [Gregor et al's] data demonstrates that their findings are consistent with the well-accepted paradigm of diffusion-based patterning and provides further support for the notion that the Bicoid profile is decoded prior to reaching its steady state". Thus, according to these authors, constant nuclear Bicoid levels are not indicative of steady-state of the gradient itself given that cytoplasmic levels may still be changing. The small diffusion coefficient of Bicoid would then be an additional argument in favor of the necessity of a pre-steady-state decoding mechanism. If this is the case, the differences in Bicoid levels between adjascent cells would be much bigger at cleavage cycle 9 (50% instead of 10% at cycle 14), thus resolving the paradox of the high precision of the hunchback response.

In their response (Bialek et al, 2008), Gregor and colleagues reply that if cells would make a decision by reading Bicoid concentration at cycle 9, the boundary between expression domains would be 5 cells wide at stage 14 (=\sqrt{2^14/2^9}), while in reality it is only a single cell wide. While they agree that the overall gradient might not be at steady-state at these early stages, they argue that the stability of nuclear Bicoid levels is functionally highly relevant given that Bicoid is a transcription factor. Finally, they also point out that the deduced local diffusion constant is so small that it is in fact incompatible with observing any Bicoid in the middle of the embryo in the first place, thus suggesting the existence of additional mechanisms to explain establishment of the gradient at the scale of the entire embryo. These and some additional arguments lead Bialek et al to conclude that "the small values of the diffusion constant for Bcd we reported are superficially consistent with their model, but the model provides no basis for understanding any of our observations."

Mmmmh... not an easy one. Those who have additional insights into these subtle but fascinating questions, please let us know!

Here is the answer provided by Kevin Struhl in an interesting interview published a few days ago in Current Biology (Current Biology 2008 18:R7):

"I think it is flawed at several levels. Anonymous reviews assume that reviewers are unbiased, objective and without personal or scientific conflicts of interest; this is not always true, especially in competitive situations, and there is no mechanism to detect such problems. Aside from the potential for abuse, anonymous reviews create an inequality between authors and reviewers that is unfair and scientifically unjustified. At many journals, particularly those run by commercial companies as opposed to scientific societies, disagreements between authors and reviewers are often adjudicated by editors with modest scientific accomplishments and experience. I favor a process in which editorial decisions are made by practising scientific experts, reviewers are identified by name, and the signed reviews and author responses published online along with the paper. Lastly, it is unfortunate that the biology community has permitted commercial companies to control most of the journals. Competition among journals and business-related decisions about scientific publishing has seriously distorted the literature, and it has created an artificial rating system that is used to judge decisions about funding and career advancement."

I highly recommend to visit the NIH VideoCasting page, which hosts many interesting video/podcasts. Even if I realize that this is a bit old according to the blogosphere time scale, I would like to point to this one: "The Future: Consumer Health Information Technology", featuring talks given at a NCI-sponsored meeting on Dec 10, 2007 by Adam Bosworth (formerly "Google Health architect", now starting his own company Keas), Bern Shen (Intel) and Bill Crounse (Microsoft).

In his introduction to the meeting, Bradford Hesse (NCI) colorfully summarizes one of the main concepts exposed by the speakers (the video is very long, so I give some pointers: 0h16min43sec) by comparing the future of healthcare to...an "IKEA flat pack": patients will progressively be empowered to assemble their own care from home, like they would build a piece of (cheap) furniture.

Adam Bosworth (0h25min53sec) presents his very pragmatic vision of how IT could concretely help healthcare (0h39min07sec): a) help the consumer to own and control his personal health data, and this already for very simple basic information; b) provide tools for doctors so that they can deliver personalized care as easily as producing a spreadsheet; c) develop tools for researchers to facilitate the design and implementation of new protocols and clinical trials.

Bill Crounse (Microsoft's other Bill...1h14min30sec) sees 5 major current trends that will increasingly challenge the healthcare system and call for IT solutions (1h26min22sec): a) increasing personal responsibility ("the end of health insurance"); b) progressive "retailization" of healthcare services (eg appearance of "retail minute clinics"); c) commoditization of healthcare providers; d) globalization of access to information (through the web of course); e) globalization of healthcare services. I recommend his little funny anecdote on the high-tech GPS wireless-connected plumber (1h25min30sec) who appears to better equipped than any practicing physician...

The speakers also all insist on the need for massive data integration promoted by the interoperability of formats and coding information, themes that probably sound familiar to many systems biologists.

Toward the end of his talk (1h35min00sec), Bill Crounse shows a short "science-fiction" movie on Microsoft's vision of the future of healthcare: a world full of credit-card sized tablet PCs, touch screens and many other very exciting gadgets (I love gadgets!). But I can't help missing a bit the warmth of human-to-human interactions within this jungle of virtual consultations, retail clinics, remote controlled metabolic parameters, etc... and I didn't quite see in that movie that the doctor would spend more time with his patient or the daughter with her sick Grandma. But this may of course only reflect some old-fashioned side of my temperament...