Synthetic gene circuits with a memory!

Imagine having a USB port in the body that we could use to insert a "flash drive" and transfer genetic data, therapies, or monitoring devices. The flash drive would have to be some kind of removable biological entity that has no problem getting in and out of the body. If you think about it, bacteria are the perfect candidates to be such devices. So, what if bacteria could be used as storage for genetic memory?

This is not so far-fetched if you think that recent studies have shown for example that genes expressed by bacteria in our guts can affect our propensity to be lean or fat. Bacteria have genes that "record" and "affect" what's going on in our body. The question is: can we control them?

Bacteria have a way of turning "on" or "off" their genes based on stimuli from the environment. Synthetic biology studies ways of using these "switches" to make "gene circuits". Genetic regulatory circuits are the biological analog of electric circuits, where genes, instead of light bulbs, are being turned on or off (by activating other genes).

Genetic circuits have numerous applications in medicine. For example, Auslaender et al. [1] used synthetic biology to create a pH sensor for cells. The researchers then implanted these cells into mice and used it as a device to detect diabetes. Lack of insulin causes an excess of acidity in the blood, and the pH drops below 7.35. Changes in pH induced by diabetes were quickly detected by the pH-sensor cells in the implanted mice. The pH information was processed and triggered a transgene expression response that resulted in the secretion of alkaline phosphatase to counteract the acidity. Basically, what the cells were able to do in the mice is: (1) detect the drop in pH; (2) trigger a response to restore the pH to normal levels.

In an electrical circuit you assemble elements like resistance and capacity. In a genetic circuit you assemble genes and "operators" able to edit the DNA in order to activate or deactivate the genes. One of such "editors" is a class of enzymes called recombinases. Apparently, there aren't many of these enzymes available, which limits the number of gene circuits one can make.

A recent study published in Science [2], however, presented a new class of such enzymes, derived from the bacteriophage Lambda, which is a virus that infects Escherichia coli. The novelty of the method doesn't stop here. You see, the goal is not just to have a working circuit, but to also make it autonomous. In other words, ideally, one wants a system able to detect responses and readjust the output based on the input it receives. The researchers devised genetic regulatory circuits able to "write", "input" and "read" genetic information.

Farzadfard and Lu [2] "converted genomic DNA into a 'tape recorder' for memorizing information in living cell populations." Their circuit, named SCRIBE (Synthetic Cellular Recorders Integrating Biological Events), responds to gene regulatory signals by generating single-stranded (ssDNA). The ssDNA is then coexpressed with a recombinase and introduces specific mutations in targeted positions of the cell DNA. The fraction of cells in the bacterial culture that carry the introduced mutations represents the biological memory at the population level.

For example, when the researchers exposed the cultures to an exposure input for 12 days (the equivalent of 120 generations in the bacterial population), they found that the

"frequency of mutants in these populations was linearly related to the total exposure time. Furthermore, we demonstrate that SCRIBE-induced mutations can be written and erased and can be used to record multiple inputs across the distributed genomic DNA of bacterial populations [2]."

It's a 'collective memory' embedded in the observed frequency of the mutation in the bacterial population. And the applications are almost infinite. I truly can't wait to see where this kind of research will take us in the future.

[1] Ausländer D, Ausländer S, Charpin-El Hamri G, Sedlmayer F, Müller M, Frey O, Hierlemann A, Stelling J, & Fussenegger M (2014). A synthetic multifunctional mammalian pH sensor and CO2 transgene-control device. Molecular cell, 55 (3), 397-408 PMID: 25018017

[2] Farzadfard, F., & Lu, T. (2014). Genomically encoded analog memory with precise in vivo DNA writing in living cell populations Science, 346 (6211), 1256272-1256272 DOI: 10.1126/science.1256272

Mitochondria to the rescue

Yes, I confess I'm quite fascinated by mitochondria. Not only their well functioning seems to be correlated to lifespan, like I discussed last time, but it's also implicated in cancer.

Briefly, last post taught us that mitochondria provide energy to the cell by producing ATP through four different oxidative complexes. However, mitochondria's oxidative activity wanes with age. Researchers found one pathway in particular that is activated in low-fat diets and high-exercise regimens, which can reverse the decrease in oxidative activity.

In 1926, a German physician named Otto Warburg discovered that, contrary to healthy cells, which produce ATP through the mitochondria oxidative complexes, cancer cells produce most of their ATP through a process called glycolisis. Glycolisis can be thought of, in lay-man terms, as fermentation of sugar. Thanks to this discover, which was confirmed across many different lines of cancer cells, Warburg was awarded the Nobel Prize in 1931. Warburg hypothesized that the underlying cause of cancer was a dysfunction in the mitochondria that led to upregulation of glycolysis.

If glycolysis is a hallmark of cancer, can it be used to target cancer cells and destroy them, while leaving the healthy cells untouched? Furthermore, can we "cure" cancer cells by restoring the mitochondria oxidative complexes?

The answer to the second question appears to be no: while it is true that mitochondrial activity slows down in cancer cells, this is not always due to mitochondrial dysfunction, rather, to disruption in signaling pathways that regulate glucose uptake and production. As the word suggests, the oxidative complexes in the mitochondria produce ATP using oxygen, whereas glycolysis produces ATP without the use of oxygen. The upregulation of glycolysis could be an adaptation of tumor cells due to their fast proliferation. Healthy cells receive oxygen through blood vessels. However, tumor cells outgrow the production of new vessels and therefore, in order to survive, they have to adapt to the absence of oxygen. Normally the absence of oxygen would lead to cell death, which is regulated by the p53 protein. As it turns out, p53 is either mutated or downregulated in tumor cells.

So, what does the Cell paper on aging teach us about cancer?

Remember that when it comes to cells, there's never an on/off switch, but rather a cascade of signals, i.e. chemicals that activate one another sort of like in a domino effect--what we call a "pathway." To reconstruct a pathway you have to look at each domino piece and how they interact with one another. That's why things get a bit complicated.

The upregulation of glycoysis happens through a protein called hypoxia-inducible factor-1, or HIF-1alpha. HIF-1alpha is a transcription factor, in other words, a protein that binds to DNA and regulates the expression of certain genes. Gomes et al. [1] found that HIF-1alpha induces some kind of metabolic reprogramming, not just in cancer cells, but also in normal tissue as a consequence of aging. Previous studies have shown that a high-fat diet increases levels of HIF-1alpha in the liver.

In [1], Gomes et al. induced a decline of mitochondrial activity in mice by knocking out the SIRT1 gene, a gene that codes for a protein called Sirtuin 1. It turns out, without SIRT1, not only did the researchers see the decline in mitochondrially encoded oxidative complexes, but they also observed high levels of HIF-1alpha and high expression levels of the genes targeted by HIF-1alpha. Gomes et al. restored expression of SIRT1 using a molecule called NAD+, thus restoring mitochondrial activity and lowering again the levels of HIF-1alpha. It would be interesting to see if this molecule could be used in cancerous cells as well, and if downregulating glycolysis would eventually kill the cancer cell given that they have to grow in a low-oxygen environment.

[1] Gomes AP, Price NL, Ling AJ, Moslehi JJ, Montgomery MK, Rajman L, White JP, Teodoro JS, Wrann CD, Hubbard BP, Mercken EM, Palmeira CM, de Cabo R, Rolo AP, Turner N, Bell EL, & Sinclair DA (2013). Declining NAD(+) Induces a Pseudohypoxic State Disrupting Nuclear-Mitochondrial Communication during Aging. Cell, 155 (7), 1624-38 PMID: 24360282

The secret to a long life? Active mitochondria!

For quite a while now we've known that if we want to live a long, healthy life, we must exercise regularly and be good about what we eat. Recent studies have added another piece to the equation: maintain mitochondrial function.

Mitochondria are organelles found in every cell of our body. They hold a very important function: they provide energy to the cell. Most cellular processes take place using energy stored in a molecule called adenosine triphosphate, or ATP, and most of a cell's supply of ATP is produced in the mitochondria through a process called oxidative phosphorylation. Mitochondria are also the only place outside the nucleus where you can find DNA: human mitochondrial DNA (mtDNA) is circular, and it contains 37 genes. Contrary to nuclear DNA, mitochondrial DNA is not unique to every individual because it is inherited from the mother's side only and, therefore, does not undergo parental genetic recombination.

How do mitochondria fit in the longevity puzzle? Lanza et al. [1] found a progressive decline in mitochondrial DNA abundance in skeletal muscle cells with age. The progressive decline of mitochondrial activity in muscular tissue implies less ATP synthesis, and, therefore, less energy for the cell. In addition, mitochondria play a role in regulating programmed cell death, "a vital mechanism to regulate development, cell numbers, and prevent the accumulation perilous tumor cells." Therefore, it is possible that mitochondria influence the loss of muscular mass associated with aging through upregulation of apoptotic processes. In their review, Lanza and Nair [1] cite studies that have shown that mitochondrial activity is reduced in older adults, though it seems to be preserved across similar activity levels, implying that exercise can slow down and even prevent this progressive loss.

"Mitochondrial DNA copy number decreases with age, which could account for the reduction of mitochondrial gene transcripts and therefore, the proteins encoded by these genes [1]."

Even though it's not clear whether the decline in mitochondrial function is a cause or a consequence of the senile phenotype, there have been some new studies suggesting that mitochondria play a major role in regulating cellular aging, and that restoring mitochondrial function can indeed slow down the aging process.

To understand why this is the case, let's go back to mitochondria's main function: they synthesize ATP through oxidative phosphorylation. Most proteins involved in this process are encoded in the nucleus, though 13 are encoded by genes in the mitochondrial DNA. This implies that in order for oxidative phosphorylation to take place and ATP be produced, the nucleus and the mitochondria have to work together and communicate closely. As we age and lose mitochondrial function, this close network weakens, causing loss of oxidative capacity.

Researchers from Harvard Medical School noticed that though there are 4 different oxidative phosphorylation complexes, the one encoded by exclusively nuclear genes does not decline with age, while the others do. Therefore, they hypothesized that the progressive decline of oxidative activity was due to a decline in mitochondrially encoded genes. This study, a joint project between Harvard Medical School, the National Institute on Aging, and the University of New South Wales, Sydney, Australia, was published recently in Cell [2]. In the paper, the authors describe a pathway that regulates mitochondria activity in skeletal muscle cells and show that, by knocking out the pathway in genetically modified mice, they could mimic aging by decreasing mitochondrially encoded oxidative phosphorylation complexes. On the other hand:

"Current dogma is that aging is irreversible. Our data show that 1 week of treatment with a compound that boosts NAD+ levels is sufficient to restore the mitochondrial homeostasis and key biochemical markers of muscle health in a 22-month-old mouse to levels similar to a 6-month-old mouse [2]."

The NAD+ compound the Harvard researchers talk about in their paper is a coenzyme that restores communication between the nucleus and the mitochondria. When levels of mitochondrially encoded mRNA are restored, ensuring that the production of mitochondrial proteins participating in the oxidative phosphorylation complexes is no longer declining, the pathways associated with low-fat diets and high exercise regimens are once again activated.

"All of the main players in the nuclear NAD+-SIRT1-HIF-1a-OXPHOS [oxidative phosphorylation] pathway are present in lower eukaryotes, indicating that the pathway evolved early in life’s history. This pathway may have evolved to coordinate nuclear-mitochondrial synchrony in response to changes in energy supplies and oxygen levels, and its decline may be a conserved cause of aging [2]."

Even more remarkable is that the pathway the researchers found is implicated in cancer tissues, too. So, while it's worth reminding ourselves that aging is NOT a disease (I hate it when I see commercials that tell me they found a "cure" for wrinkles!), there are many age-related diseases, including cancer, that could benefit from these findings. As always, it remains to be seen whether the mouse model is reproducible in higher mammals, but finding and understanding these pathways is indeed a great step forward.

[1] Lanza IR, & Nair KS (2010). Mitochondrial function as a determinant of life span. Pflugers Archiv : European journal of physiology, 459 (2), 277-89 PMID: 19756719

[2] Gomes AP, Price NL, Ling AJ, Moslehi JJ, Montgomery MK, Rajman L, White JP, Teodoro JS, Wrann CD, Hubbard BP, Mercken EM, Palmeira CM, de Cabo R, Rolo AP, Turner N, Bell EL, & Sinclair DA (2013). Declining NAD(+) Induces a Pseudohypoxic State Disrupting Nuclear-Mitochondrial Communication during Aging. Cell, 155 (7), 1624-38 PMID: 24360282

Cancer-killing viruses

We learned last time that cancer cells are cells whose DNA has been damaged beyond repair. Somatic mutations have accumulated to the point that the cell regulatory mechanisms no longer function, causing uncontrolled growth and proliferation. Despite being anomalous, cancer cells are still part of what the immune system recognizes as "self", which makes finding a cure for cancer such a hurdle. Therapy, when available, is often invasive and debilitating because the only way to make sure that all cancer cells in the body are destroyed is to stop all cells, even healthy ones, from growing. Drugs targeted at the tumor tissue only are a good alternative, though they still need to be perfected. Another way to overcome the hurdles is to train our immune system to recognize cancer cells and destroy them. In the past, I've discussed ways to do this through gene therapy and cancer vaccines.

So when my friend Mike Martin sent me this story, I thought, "Nice. Another cancer vaccine success story." As I read through, though, I realized that this wasn't quite a vaccine. It was a deadly virus turned into a "good" virus.

This is the story of the "redemption" of the poliovirus. :-)

Viruses hijack cell machinery (proteins) in order to reproduce. They do so because first of all, they are very small and they can't possibly package all the proteins they need into their tiny shell. Also, by using the cell's proteins instead of viral ones they disguise themselves: less viral proteins means more chances to evade the host immune system. When successful, most viruses end up killing their host cell.

What if we could do the opposite? What if we could hijack the viral proteins, instead, and use their "killing" machinery to ... kill cancer cells? That's the brilliant idea Dr. Matthias Gromeier, from Duke University had, and the basis of his research on oncolytic viral immunotherapy.

An oncolytic virus is a virus that targets cancerous cells. The term was coined after reports of cancer remissions that coincided with a viral infection or a vaccination. While in vitro models had originally given good results, the in vivo use of oncolytic viruses has shown to be more challenging than originally anticipated due to the complicated relationship between a virus and its host. One thing that makes the immune system so fascinating and yet so complicated to study, is that it depends not only on genetics ("innate immunity", the immunity we are born with), but also on "experiences" and "exposures" ("acquired immunity," the immunity that results from exposure to pathogens and immunogens throughout our lifetime), which are often much harder to reconstruct and fold into a model. So, whenever you try to use a virus for therapy, as in viral vectors for gene therapy, for example, you face the obstacle of different immune systems, some of which may have encountered the virus (or a similar one) before and will promptly destroy it.

In a 2011 paper [1], Gromeier and his group described PVSRIPO, a prototype nonpathogenic poliovirus they designed to treat glioblastoma, one of the most common and most aggressive brain tumors. The prototype is a poliovirus recombinant engineered to replicate exclusively in malignant cells. It targets one protein in particular, Necl-5, a tumor antigen expressed by many tumor cells. Think of it as a red flag that the tumor cells carry. PVSRIPO is able to "see" the red flag and attack the cell, eliciting "efficient cell killing and secondary, host-mediated inflammatory responses directed against the infected tumor [1]." In other words, not only it kills the cell, it also elicits immune responses against the affected area.

The prototype has been FDA-approved and is currently being tested in clinical trials with patients with glioblastoma multiforme, though it already made news:

"Of the seven others who later enrolled in Dr. DesJardins' clinical trial, one patient responded like Lipscomb [whose brain tumor is shrinking and has survived cancer for a year and a half, four times longer than most people with her type of tumor]. Two patients, whose immune systems were already severely damaged, did not. It’s too early to tell with the remaining three patients, but animal studies suggest that once the body recognizes and destroys the tumor, it won’t return. If those results hold up, researchers hope to apply the same technique to a whole range of other cancers, including melanoma and prostate cancer [2]."

[1] Christian Goetz, Elena Dobrikova, Mayya Shveygert, Mikhail Dobrikov & Matthias Gromeier (2011). Oncolytic poliovirus against malignant glioma Future Virology DOI: 10.2217/fvl.11.76

Because this makes me smile every time I watch it: the inner life of a cell

I can't remember if I've already shared this video here, but if I have, it's worth seeing more than once. In fact, I watch it every time I get frustrated at work. Every so often we get caught up in failed experiments, dead calculations, politics, grants, etc., and we forget why we are doing this: because deeply inside there's the mysterious, magical beauty of what makes life possible: the cell.

"Created by XVIVO, a scientific animation company near Hartford, CT, the animation illustrates unseen molecular mechanisms and the ones they trigger, specifically how white blood cells sense and respond to their surroundings and external stimuli."

We read about all these mechanisms in textbook, but this video brings them to life, showing you the dynamics, the landscapes, the interactions. It wows me every time. You can read the full story about the video here.

Towards a new era of synthetic genetics

NOTE: this is a short post since this particular study has already been extensively discussed all over the scientific blogosphere, see for example this post. Here I just want to give a very general overview for those who may have not yet heard about this. I think my writer friends in particular will be extremely intrigued by this news.

In life as we know it, the storage and propagation of genetic information relies on two molecules: RNA and DNA. Both are made of building blocks called nucleotides, which are in turn composed of a nitrogenous base, a five-carbon sugar, and one phosphate group. The sugar in the RNA nucleotide is called ribose, and the one in the DNA nucleotides deoxyribose.

Most likely, the very first forms of life had RNA only. Being a single stranded molecule, it is less complex than DNA, but also less stable. As life became more complex, it evolved towards a more complex molecule. But have RNA and DNA always been the only two existing molecules capable of heredity and evolution?

In order to address the question, Pinheiro et al. [1] studied six new molecules (XNAs, for xeno nucleic acids) with the capability to store and propagate genetic information. The molecules were obtained using alternative sugar-like components in lieu of the five-carbon sugar. Synthetic nucleic acids are only the starting point. The key point the researchers had to address was: can these be synthesized back and forth from DNA? Because you see, the way genetic information is stored and passed on is through back and forth transcription between DNA and RNA so that proteins can be made.

Indeed, Pinheiro et al. engineered special polymerase enzymes able to do exactly that: reverse transcribe XNA into DNA and, viceversa, forward transcribe DNA into XNA.

"All six XNAs studied by Pinheiro et al. bind to complementary RNA and DNA and are resistant to degradation by biological nucleases. Construction of genetic systems based on alternative chemical platforms may ultimately lead to the synthesis of novel forms of life [2]."

I can see the imagination of a few people out there running wild. Yes, it is indeed wild. Think of the possibilities. Pinheiro and colleagues call this field of synthetic genetics a "route to novel sequence space."

Of course, this is just a first step. Let's not forget that life as we know it took billions of years to evolve from those first molecules of RNA. In my previous post I quoted Waddington's metaphor who compared the evolution and diversification of cells to marbles rolling down a rugged landscape. Gravity is the driving force and the pits and inclines are the constraints. What is to happen, though, if we, humans, start changing this landscape?

[1] Pinheiro, V., Taylor, A., Cozens, C., Abramov, M., Renders, M., Zhang, S., Chaput, J., Wengel, J., Peak-Chew, S., McLaughlin, S., Herdewijn, P., & Holliger, P. (2012). Synthetic Genetic Polymers Capable of Heredity and Evolution Science, 336 (6079), 341-344 DOI: 10.1126/science.1217622

[2] Joyce, G. (2012). Toward an Alternative Biology Science, 336 (6079), 307-308 DOI: 10.1126/science.1221724

What better way to start the new year than... with a carnival?

...and not just any carnival: the Molecular Biology Carnival!

Happy New Year, everyone! I'm starting off the new year by hosting the January edition of the Molecular Biology Carnival -- what a great honor! And a very special edition indeed, with lots of my favorite things: vaccines, viruses, proteins, bacteria, and more!

Let's start off with James Byrne's holiday-themed post (Merry) Christmas Disease, where the ailment is not some kind of viral disease you might catch on Christmas eve, but a rare form of hemophilia named after a patient diagnosed with it, Mr. Stephen Christmas.

We all know vaccines are delivered through viral vectors, but what about... parasites? Zoonotica explains how a group of researchers genetically modified a trypanosome parasite to vaccinate cattle from redwater fever.

Talking about parasites, did you know that viruses have their own share, too? These "viral" parasites are called virophages, and they can only reproduce in cells that have already been infected by a helper virus. In his post Virophages and the evolution of transposable elements, Habib Maroon discusses a new virophage recently discovered, called Mavirus, which amazingly sheds light on the origin of an intriguing DNA element, the transposons (yes, the "jumping genes" I've covered in an earlier post).

Let's stick with viruses and ask the following question: how come some viruses are degraded by our immune system's sentinels, the T-cells and B-cells, and others, like measles and HIV instead have developed a mechanism to infect those very same cells that are supposed to destroy them? Connor Bamford, in his post Viruses at the crossroads of infection looks at a paper that suggests the answer may be hidden in the virus's sugar coating.

And in another great post from his Rule of 6ix blog, Connor talks about the influenza virus and dendritic cells: immune response or Trojan horse?

From viruses to bacteria and, in particular, bacterial genes: Gemma Atkinson, in her post Bacterial genes in eukaryotes - function and phylogeny presents two papers that look at bacterial genes in eukaryotes.

Still from the amazing world of bacteria, S. E. Gould reports of a new group of magnetic bacteria, better known as magnetotactic bacteria. As S.E. Gould explains, these organisms "contain small nanoparticles of magnetic material which allow them to swim along magnetic field lines." How cool is that?

And yet another bacteria marvel: off the coast of Costa Rica, white and hairy crabs known as Yeti crabs farm bacteria on their claws, as Lucas Brouwers explains in his post Yeti Crabs grow bacteria on their hairy claws.

Finally, DNA Testing presents DNA Paternity Testing on the Rise posted at DNA Testing Blog - DNA Paternity, Sibling, & Biological Family Testing.

That's all for this edition. Don't forget to submit your entry for the next edition of the molbio carnival using the carnival submission form. Past posts and future hosts can be found on the blog carnival index page.

Photo: "Beam me up, Spock!" A.K.A.: a "zoom blur" (zoom while the shutter's open) of a decorated tree in downtown.