Fifth in a series of posts inspired by ten topics in ‘Insights of the Decade’ from the December 17, 2010 special issue of Science Magazine The topics are: Inflammation, climatology, tricks of light, alien planets, the microbiome, cell development, Martian water, the DNA time machine, cosmology and epigenetics. The original articles are now behind a paywall; they won’t be reproduced here, but their gist is present. I’ll try to put them in context and specifically within the Impact Areas of SciTechStory.

When I was a kid (a few decades ago), my parents took me to Yellowstone National Park (Wyoming, USA). I remember marveling at many things, not the least of which were the brilliant colors in and near the pools of very hot water – oranges, yellows, greens – in all hues. I wondered aloud what made the colors and I was told the common wisdom of the day that most of the colors were from minerals dissolved in the water, except maybe some green, which was from algae. There were probably a few biologists of the time who suspected the presence of bacteria in these pools, but it was generally agreed that bacteria couldn’t live in the heat of these waters.

The concept of the thermophile, heat loving microorganisms, was probably first used in 1879 and promptly put on the mental back-shelf by almost all biologists. While it was accepted that some forms of plant life (algae) could tolerate higher temperatures (between 60 and 80 degrees Celsius, 140-180 degrees Fahrenheit), it just didn’t seem likely that any form of animal life could survive. This ‘intuition’ was generally frozen into many scientists’ thinking in the form of a bias.

That bias persisted until research in the 1980’s and 90’s showed that not only could bacteria live – and thrive – in hot environments, but there were, in fact, such a thing as hyperthermophiles that could live in water up to and above the boiling point. Many of these microorganisms were highly specialized bacteria, and many more were from what is now recognized as a completely distinct domain of life – the Archaea. It is such creatures that produce the colorful bands around thermal pools in Yellowstone. These days, the study of such extremophiles, life in extreme conditions, is of enormous biological and even commercial importance.

In a similar vein, scientists have known since the 19th century that the human body contained bacteria. Mostly though, these bacteria were known as the cause of disease. It was assumed that they didn’t belong in the body. This intuition became frozen into common knowledge, a bias. Even when it was later shown that some bacteria lived inside the body, particularly in the gut and were probably part of the digestive system, the focus was still very much on dealing with the destructive bacterial invaders. In short, the bias continued to guide the accepted wisdom and most of the research effort.

Unbelievable as it might seem now, it wasn’t until the year 2000, when Nobelist Joshua Lederberg published in Science a broadside aimed at his fellow bacteriologists:

New strategies and tactics for countering pathogens will be uncovered by finding and exploiting innovations that evolved within other species in defense against infection. But our most sophisticated leap would be to drop the Manichaean view of microbes – “We good; they evil.” Microbes indeed have a knack for making us ill, killing us, and even recycling our remains to the geosphere. But in the long run microbes have a shared interest in their hosts’ survival: A dead host is a dead end for most invaders too. Domesticating the host is the better long-term strategy for pathogens.
[Source: From Science Magazine: Infectious History]

Even Lederberg couldn’t purge his language of the ‘pathogen’ view of microorganisms, but those who heeded his call for a more equanimous view of microbes in the human body soon began to discover that the reality called for a wholly different view. For example, as is now often cited, the human body contains far more cells of microbes than our own cells – by almost 10 to 1. There are at least 1,000 species living inside us, and most of them are symbiotic or at least commensal (one sided benefit). This vast community of mostly non-pathogenic microorganisms is now called the microbiome, an ecology inside us.

As you can imagine, with only ten years of research microbiologists have just begun to scratch the surface of this newly appreciated relationship between human and microbe. For example, while scientists knew that bacteria were involved with digestion, a 2004 study by a team working with Jeffrey Gordon at the Washington University School of Medicine (St. Louis, Missouri, USA) discovered that bacteria were essential partners in helping to break down the food we eat and to make metabolic use of it.

The microbiome is now linked to various aspects of human health, including the proper functioning of our immune system. It is also suspected that bacterial DNA may play an active role in our own DNA development. With each new study the links to bacteria within the body proliferate. This new view is even extending to diminish an even greater bias – the bias against viruses. A new term, the virome was coined to encompass the role that viruses have within the normal functioning of the body, as strange as that may seem.

Some of the practical impact of research into the microbiome and virome will be a better understanding of their benefits to the body – and how to keep it that way. Scientists are also learning that the microbiome is highly individualized; we can be identified as individuals by the denizens of our gut. These same fellow-travelers also identify our culture and genetic background. This has led research into areas of diet and body chemistry. In fact, the knowledge of the microbiome is leading research into fundamental metabolic pathways that were not even suspected ten years ago. This work will in time have an impact on cell biology, immune systems, and genetics.

It should also be a good time to draw a deep breath, assemble some patience, and wait for the dust to settle. There is very likely to be two kinds of news on this story: What Venter and team actually accomplished, and what the world makes of it. The former will probably be less (and more) than most people think it is. The latter will be in the larger banner headlines, mostly with worries and condemnations.

That said, this is big news, even if it turns out to be just the first movements of a baby step. In the just released article in Science Express, Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome the scientists describe the step by step process of creating a bacterial chromosome, the successful transfer of that chromosome into another bacterium where it replaced the native DNA, and how – with that synthetic genome – the bacterium began replicating its new DNA and creating new proteins. In short, it was alive.

Now even to a casual reader, the idea of putting the synthetic DNA into another (already existing) organism – sort of like changing the blueprints for a work in progress – isn’t the same as building life from scratch. There are a lot of organic shortcuts involved. Still, it is equally obvious that building DNA synthetically that actually works is no small accomplishment.

This project began around 1995, when Venter and other colleagues began looking for the bacterium with the smallest genome, which they found in Mycoplasma genitalium, a microbe with only 500 genes. Theoretically fewer genes would make it easier to synthesize the DNA, but it took more than a decade. By 2007 the Venter team could demonstrate that it was possible to move the (natural) DNA of one bacterium into another bacterium. In 2008 they announced the creation of an artificial “watermark,” a genetic code embedded in the DNA that would instantly identify anything created with that DNA as synthetic.

Unfortunately, the bacterium M. genitalium was such a slow breeder that the scientists were forced to abandon it as their target. They switched to M. mycoides a much faster growing bacterium. By mid-2009 they were able to demonstrate the transfer of DNA from M. mycoides to another bacterium, M. capricolum, a close relative. The next step was to build synthetic DNA.

Yeast was used as the assembly ground for the new DNA, where the 10,000 genetic sequences were stitched together – mostly one by one – until a complete genome was finished. They transplanted this synthetic DNA into M. capricolum. Nothing happened. It was probably a ‘bug’ in the code, a lot like software. Finding the mistake took three months.

More months passed while they tried many genomic transplants. Finally, in early 2010, they found a genome combination that worked: M. capricolum was making proteins as if it was M. mycoides. One cell was transformed into another.

You can call that creating life, or not. It’s arguable – and it will be argued, no doubt about it. At the very least, the part of the argument about whether this is scientifically solid ‘creation of life’ or some kind of preliminary or intermediary step could be interesting and possibly bear fruit. Other kinds of arguments…they are also important, but will probably produce far more heat than light. It will be interesting to see what cultural impact this has.

In a piece of world-class timing, the horror movie Splice will be released June 4. I’m using the word ‘horror movie’ instead of science fiction movie, because while what is presented in the movie is still science fiction – with today’s news, it’s not so much science fiction any more. The movie features a couple of good actors in Adrien Brody (The Pianist) and Sarah Polley (Away from Her). Word on the street is that it’s a pretty good movie. If so, it will find itself in a maelstrom of interest. Since it presents the work of geneticists going amok (the geneticists and their creation), it will be instantly seen as presaging the dangers of doing the kind of work the Venter team – and many other scientists – have been pursuing for at least a decade. I love it when art reflects life and life reflects art…in more ways than one.

More fundamentally, perhaps, will be the impact synthetic life – not just this first step, but the implications of what can be done in the future – will have on religious beliefs. It also has almost immediate practical implications for ethics, and no doubt government regulation. Beyond that, as the technology ramps up…what a phrase, creating life forms…as the technology ramps up, this will have economic and legal ramifications as well. Venter’s group is already applying for patents.

Until now, scientists believed these pathways operated largely independently of one another to produce protein signals that travelled to the nuclei of the embryo’s cells where DNA is stored. There, coordination of these signals was thought to occur when they interacted with cell DNA to influence and control the expression of genes. Results published March 9 in the journal Current Biology, however, suggest that competition for the MAPK enzyme among proteins in different pathways influences which signals are sent to cells, establishing a biochemical mode of signal integration that adds a previously unrecognized layer of complexity and control to embryonic development [emphasis added].

[Source: Cell News]

The study, led by a team from Princeton University (New Jersey, USA), was researching the chains of chemical reactions – chemical pathways – that lead undifferentiated cells (stem cells) to the correct location, to develop the necessary characteristics, so that an embryo develops the appropriate organs and the correct form for its species. Most of this process, guided by what is called the regulated gene expression network, is studied by systems biology and at the molecular level is barely charted territory. (This also means there’s more chance for the unexpected.)

Specifically, the research team was studying the chemical pathways of protein formation, the building blocks of living tissue, and how they integrate different signals (chemical states) that direct early embryonic development. As the quote points out, it was believed that these pathways were separate, and communicated only with DNA in the nucleus of cells to receive instructions for development. The focus of attention was a particular enzyme (a chemical that enables or speeds-up reactions) known as MAPK (mitogen-activated protein kinase). The MAPK enzyme is found in all complex organisms, and appears in chemical networks that are critical for cell development.

The subject for their observations was the old lab-buddy the fruit fly (Drosophila melanogaster), whose embryos were used to study development. What they discovered was that different protein pathways competed for the MAPK enzyme. The competition didn’t necessarily have ‘winners’ and ‘losers’, but it was a competition for scarce resources, so that if one protein pathway was (for a time) more successful in acquiring MAPK, then whatever it was building would grow faster. This competition is a control mechanism. While the DNA may be making the blueprint, this control mechanism (or others like it) may determine the distribution of resources determining what grows where and when. The idea that proteins working together – even outside the instructions of DNA – may be guiding embryonic development is…unexpected. For embryology it could be revolutionary, although that overworked word should take on meaning only after a lot more testing and analyzing the findings.

The research team was able to track the effect of this competition for the MAPK enzyme. For example, the portion of the embryo that would become the fly’s head was where the concentration of protein from one pathway was high. That’s where MAPK would also be present in higher concentration than in another pathway protein, say at the tail of the fly.

Further work: Just two words, but in this case it’s like opening a dictionary, which you thought had a thousand pages but now has many thousand pages. Having broached the concept that protein pathways can produce signals that compete for enzyme (and possibly other) resources, and that this competition is yet another complication to the network of gene expression during embryo development…to a molecular biologist this might suggest all kinds of questions (experiments). How do the proteins compete? What determines how long one protein is more successful than another? Are there other enzymes that have similar relationships with signaling pathways? Is this same effect at work in other species? Many, many questions – with the potential to edit (if not re-write) the book on developmental biology.

In some ways, this suddenly expanding field of research sounds familiar – it echoes the discovery that within cells epigenetics (gene expression directed by something other than DNA, especially proteins) is much more complex and influential than originally thought. These are heady discoveries that quicken the pulse of veteran biologists and make PhD candidates salivate over dissertation topics. Much further work indeed.

The work was undertaken by the European Union funded MetaHIT (Metagenomics of the Human Intestinal Tract) project with collaborating research teams in Europe and China. [Source: Nature Magazine: A human gut microbial gene catalogue established by metagenomics sequencing] The scope of the project, the vast amount of data it processed, and its potential impact make this something of a landmark study. It seems that everyone has their favorite (sort of weird science) statistics and information from this study. Here’s a sample:

- 576.6 gigabases of gene sequence
- 3.3 million non-redundant microbial genes
- The human body hosts trillions of micro-organisms, most of which live in our gut
- There are more bacterial cells in our body than our own cells (however, our cells outweigh them)
- 99% of the genes are bacterial, from about 1,150 species
- About 160 species of gut bacteria are shared by all people
- You have lots of co-workers in your gut
- Most of the microbes in the gut are not harmful (when in balance)
- Many of the microbes contribute important chemicals and processes to digestion
- Your gut may actually be telling you something

This last one – Your gut may be telling you something – is one of the more interesting spins. It’s a characterization of the possibility the bacteria in your gut produce enzymes, messenger molecules, and other chemistry that may ‘dictate’ your state of hunger (and for what) and perhaps regulate other ‘feelings’ about your health. I’m sure there will be follow-up research in this area.

However, the more important research, already begun in this study, is a result of comparing the genomic components of this microbiome between people from different locations and health conditions. The study itself involved testing the feces from 124 Europeans and is now being widened to 350 individuals with a variety of obesity and bowel problems. While it showed quite dramatically that most of the microbes in the human gut are shared by all of us, there are significant differences – sometimes by locale, sometime by individuals. Analysis of these differences, especially for medical purposes, should be some of the more important findings developed from the data in the catalog.

During much of human history shamans and doctors have studied our stercoraceous output for signs of disease (and other problems). We’ve come a long way…

It’s reasoned that a chemical compounds began reaching ‘proto-life’ conditions, one of the first of the more complex organic compounds was probably a ribozyme, or something very much like it. The question was, “How could something this basic – only five nucleotides – produce a chemical reaction typical of an enzyme?” But it does. The experimental work with RNA ribozyme showed that it does, indeed, produce reactions.

Here’s what Professor Michael Yarus (University of Colorado) found as the key point:

Yarus noted that the RNA World hypothesis was complicated by the fact that RNA molecules are hard to make. “This work shows that RNA enzymes could have been far smaller, and therefore far easier to make under primitive conditions, than anyone has expected.”

If very simple RNA molecules such as the product of the Yarus lab could have accelerated chemical reactions in Earth’s primordial stew, the chances are much greater that RNA could direct and accelerate biochemical reactions under primitive conditions.

Before the advent of RNA, most biologists believe, there was a simpler world of chemical replicators that could only make more of themselves, given the raw materials of the time, Yarus said.

“If there exists that kind of mini-catalyst, a ‘sister’ to the one we describe, the world of the replicators would also jump a long step closer and we could really feel we were closing in on the first things on Earth that could undergo Darwinian evolution,” Yarus said.

[Source: EurekAlert]

Keep in mind that the researchers are not saying they have the ‘original’ ribozyme, only that a ribozyme-like enzyme – and early form of RNA – was very likely a component of the many compounds necessary to produce a self-replicating entity (a.k.a. life). Nevertheless, this is an important step in the direction that will eventually lead to the creation of the necessary components.

Of course, knowing that implanting organs is difficult (rejection, infection, etc.), how is it that a mouse liver could accept human liver cells? The research team from the Salk Institute for Biological Studies (USA) explains it this way:

The Salk team had previously generated a mouse with a partially “humanized” liver, but wanted to improve their method to achieve almost complete transformation. They use a special mouse that has liver problems of its own, but whose problems can be kept in check with a drug called NBTC. Taking away NBTC allows human hepatocytes to take hold and populate the mouse liver with human cells.

The team perfected this system so that nearly 95% of the liver cells are of human origin, but the important question was whether they would behave like a human livers. To test this, the researchers exposed the mice to Hepatitis B and Hepatitis C and found that, unlike normal mice, which are resistant to these viruses, the chimeric animals developed the disease.

More importantly, using pegylated interferon alpha 2a-the standard treatment for Hepatitis C-the researchers showed that the “humanized” liver inside the mouse responds just like a normal human liver. The team also tested additional experimental drugs and found that they too behaved as they did in humans.

[Source: EurekAlert]

This method of testing liver cells and function through the intermediary of a mouse may not extend to other organs, but even so this is a ‘logical’ yet extraordinary application. It may be part of a growing capability to use animals of many kinds (pigs certainly jump to mind) to develop human analogous tissues and organs. Incidentally, the word for this kind of ‘guinea pig’ (test animal) is chimeric, which is ironically a lot like chimerical.

The work was conducted at the cellular level, using cells derived from heart transplant tissue. The cells were kept in vitro (Petri dishes) and subjected to stress, in particular fats known to cause inflammation and atherosclerosis.

“The genes responded differently to inflammation depending on their genetic makeup,” said first author Casey Romanoski, a UCLA graduate student in human genetics. “About 35 percent of the most affected genes were influenced by the interaction between their genetic variants and the fats.”

“You can’t effectively study genes divorced from their environment,” she added. “The missing link lies in the intersection of genes with their environment.”

[Source: EurekAlert]

If you think you’ve heard some of this before, you probably have – only from a different angle. There are many studies based on studies of people with heart disease and their personal habits that lead to conclusions such as “Obesity linked to heart disease.” These are largely statistical studies. Many other studies, more laboratory oriented statistical studies, find links between specific genes and incidences of a disease with conclusions such as “Gene found linked to heart disease.” The UCLA study is at the cellular level, with biochemistry being the principle interest, and results looking like “Body fats contribute breakdown of protein construction.”

The links between, say obesity and heart disease are more or less intuitive, but also very generalized. The UCLA study begins the task of pinning down not only the genes involved, but also the specific molecular pathways that lead from the wrong kind of body fats interacting with genes to produce the wrong kind of proteins. There is still much more detailed work yet to be done.

A team of researchers from Jason Chin’s group at the Medical Research Council Laboratory of Molecular Biology, Cambridge (UK) have succeeded with a proof of concept demonstration for codons containing four nucleotides. Before going into the details of how this was done, a question: Why is it important? The question is (or should be) asked of every experiment, but here is a case where it begs another question – If Nature only needs codons with 3-letter nucleotides, why bother with 4-letter?

For one thing, it increases the number of nucleotide combinations to 256. From the point of view of synthetic biology, that’s 192 more blocks in the kit for building proteins – creating proteins that have never been seen before…and all that might entail. More immediately, the techniques used to create a codon of four nucleotides will have great impact on proteomics (the systemic study of proteins) because it presents a new and better way to conduct the experiments.

To achieve the new approach, the research team had to solve a number of difficult points: Proteins are made in small enclosures called ribosomes within a cell. Ribosomes receive instructions for manufacturing proteins from messenger RNA (mRNA) coming from DNA in the cell nucleus. The mRNA brings the instructions, normally, in 3-letter nucleotide codons. Since the ‘new’ codons can’t be used in the normal ribosomes, the researchers had to create a new type of ribosome and a new type of mRNA. They accomplished this by inserting genes into our old friend the bacteria E. coli that caused the creation of ‘extra’ ribosomes, so called orthogonal (independent) ribosomes or o-ribosomes. These o-ribosomes exist alongside the normal ribosomes and very importantly do not interfere with the creation of normal proteins needed by the cell. The extra ribosomes were also modified genetically so that they could accept the 4-letter nucleotide codons. Likewise altered tRNA was introduced into the o-ribosome, which would accept 4-letter codons. Finally, to introduce altered codons and their amino acid instructions, the E. coli mRNA was modified.

Once the machinery was in place, the team put it to the test. A normal 3-letter anti-codon, CTT in mRNA, would specify the amino acid phenylalanine. In turn this would be the tRNA codon AAG inside the ribosome. Shifting to a 4-letter codon, they decided to make an unnatural amino acid p-azido-l-phenylalanine. This was assigned – made up, since there are no naturally occurring 4-letter codes – mRNA AGGA, tRNA UCCU. This coding they ran through the o-ribosomes to produce a mutant form of the protein calmodulin, which uses p-azido-l-phenylalanine in its construction.

In essence, the 4-letter codon and the machinery to process it is a technology. At least for now there is no direct use within human cells, but that’s not the point. This is an enabling technology, one that will make it possible for an incredible number of new experiments in protein development, which will hopefully lead to better understanding of the microbiology of proteomics and epigenetics. It will also enable ‘a new laboratory space’ for creation of synthetic proteins – the building blocks of cells and living tissue. Dr. Frankenstein never had it so fundamentally good.

One of the reasons for confidence in the eventual discovery of how life evolved and the recreation of the pathways – the ability to create life ‘from scratch’ (…create life in a test tube, as the expression goes) – is the stunningly rapid advancement of microbiology and bioengineering. More and more detail is accumulating about the properties and processes of reproduction (DNA replication), and the life-sustaining processes of living cells. This detail is no longer the raw description drawn from simple observation, but the (more or less) precise description of chemistry. We’re pursuing the detail into the molecular level; into the nanoscale. It’s a painstaking process, but as the tools (scientific equipment) and the theory improves; the flow of results is gaining momentum.

Another approach to solving the mysteries of creating life is to make a model and run it on a computer. Better still, run the model on not one computer, even a supercomputer, but perhaps thousands of computers. A recent article in the New York Times by veteran science writer John Markoff, highlighted an attempt by a scientific team to enlist the help of people with a personal computer to create a network to crunch the fantastic number of repeated (iterated) calculations necessary to mimic the effect of millions of years of evolution.

The effort, dubbed the EvoGrid, is the brainchild and doctoral dissertation topic of Bruce Damer, a Silicon Valley computer scientist who develops simulation software for NASA at a company, Digital Space, based in Santa Cruz, Calif.

Mr. Damer and his chief engineer, Peter Newman, are modeling their effort after the SETI@Home project, which was started by the Search for Extraterrestrial Intelligence, or SETI, program to make use of hundreds of thousands of Internet-connected computers in homes and offices. The project turned these small computers into a vast supercomputer by using pattern recognition software on individual computers to sift through a vast amount of data to look for evidence of faint signals from civilizations elsewhere in the cosmos.

The EvoGrid goal is to detect evidence of self-organizing behavior in computerized simulations that have been constructed to model the first emergence of life in the physical world.

[Source: New York Times]

As pointed out in the article, computer models of such complex systems are problematic. Because the models are tested in abstract terms (not in real life), there’s no guarantee that failures in assumptions or the built-in processes will be detected. Nevertheless, the exercise – if that’s all it is – may provide useful insights. It also expands our knowledge of the capabilities, and limitations, of massive computation. The system being put together for EvoGrid is interesting in its own right:

To quickly build the EvoGrid, the researchers are relying on two open-source software projects.

Boinc is a system financed by the National Science Foundation that uses the Internet to permit scientists to take advantage of free computing cycles available on network-connected computers. Last week, for example the system was composed of more than 500,000 computers that generated an average of almost 2.45 petaflops of computing power. By contrast, in June of this year, the world’s most powerful supercomputer, built by I.B.M. at Los Alamos National Laboratories, produced 1.1 petaflops.

To simulate digital evolution, the EvoGrid will use a second program, Gromacs, developed at the University of Groningen in the Netherlands, to model molecular interactions. EvoGrid researchers hope to create a computer model that replicates the early ocean and then use it as a virtual “primordial soup” to quickly evolve digital forms.

Although the human genome sequence faithfully lists (almost) every single DNA base of the roughly 3 billion bases that make up a human genome, it doesn’t tell biologists much about how its function is regulated. Now, researchers at the Salk Institute provide the first detailed map of the human epigenome, the layer of genetic control beyond the regulation inherent in the sequence of the genes themselves.

“In the past we’ve been limited to viewing small snippets of the epigenome,” says senior author Joseph Ecker, Ph.D., professor and director of the Genomic Analysis Laboratory at the Salk Institute and a member of the San Diego Epigenome Center. “Being able to study the epigenome in its entirety will lead to a better understanding of how genome function is regulated in health and disease but also how gene expression is influenced by diet and the environment.”

“This paper exemplifies the goals of the NIH Roadmap for Medical Research’s Epigenomics Program,” said Linda Birnbaum, Ph.D., director of the National Institute of Environmental Health Sciences, one of the NIH institutes funding this program. “The science has matured to a point that we can now map the epigenome of a cell. This paper documents the first complete mapping of the methylome, a subset of the entire epigenome, of 2 types of human cells – an embryonic stem cell and a human fibroblast line. This will help us better understand how a diseased cell differs from a normal cell, which will enhance our understanding of the pathways of various diseases.”

[Source: AAAS:]