Complex Logic Circuit Made from Bacterial Genes

By force of habit we tend to assume computers are made of silicon, but there is actually no necessary connection between the machine and the material. All that an engineer needs to do to make a computer is to find a way to build logic gates -- the elementary building blocks of digital computers -- in whatever material is handy.

So logic gates could theoretically be made of pipes of water, channels for billiard balls or even mazes for soldier crabs.

By comparison Tae Seok Moon's ambition, which is to build logic gates out of genes, seems eminently practical. As a postdoctoral fellow in the lab of Christopher Voigt, PhD, a synthetic biologist at the Massachusetts Institute of Technology, he recently made the largest gene (or genetic) circuit yet reported.

Moon, PhD, now an assistant professor of energy, environmental and chemical engineering in the School of Engineering & Applied Science at Washington University in St. Louis is the lead author of an article describing the project in the Oct. 7 issue of Nature. Voigt is the senior author.

The tiny circuits constructed from these gene gates and others like them may one day be components of engineered cells that will monitor and respond to their environments.

The number of tasks they could undertake is limited only by evolution and human ingenuity. Janitor bacteria might clean up pollutants, chemical-engineer bacteria pump out biofuels and miniature infection-control bacteria might bustle about killing pathogens.

How to make an AND gate out of genes

The basis of modern computers is the logic gate, a device that makes simple comparisons between the bits, the 1s and 0s, in which computers encode information. Each logic gate has multiple inputs and one output. The output of the gate depends on the inputs and the operation the gate performs.

An AND gate, for example, turns on only if all of its inputs are on. An OR gate turns on if any of its inputs are on.

Suggestively, genes are turned on or off when a transcription factor binds to a region of DNA adjacent to the gene called a promotor.

To make an AND gate out of genes, however, Moon had to find a gene whose activation is controlled by at least two molecules, not one. So only if both molecule 1 AND molecule 2 are present will the gene be turned on and translated into protein.

Such a genetic circuit had been identified in Salmonella typhimurium, the bacterium that causes food poisoning. In this circuit, the transcription factor can bind to the promotor of a gene only if a molecule called a chaperone is present. This meant the genetic circuit could form the basis of a two-input AND gate.

The circuit Moon eventually built consisted of four sensors for four different molecules that fed into three two-input AND gates. If all four molecules were present, all three AND gates turned on and the last one produced a reporter protein that fluoresced red, so that the operation of the circuit could be easily monitored.

In the future, Moon says, a synthetic bacterium with this circuit might sense four different cancer indicators and, in the presence of all four, release a tumor-killing factor.

Crosstalk and timing faults

There are huge differences, of course, between the floppy molecules that embody biological logic gates and the diodes and transistors that embody electronic ones.

Engineers designing biological circuits worry a great deal about crosstalk, or interference. If a circuit is to work properly, the molecules that make up one gate cannot bind to molecules that are part of another gate.

This is much more of a problem in a biological circuit than in an electronic circuit because the interior of a cell is a kind of soup where molecules mingle freely.

To ensure that there wouldn't be crosstalk among his AND gates, Moon mined parts for his gates from three different strains of bacteria: Shigella flexneri and Pseudomonas aeruginosa, as well as Salmonella.

Although the parts from the three different strains were already quite dissimilar, he made them even more so by subjecting them to error-prone copying cycles and screening the copies for ones that were even less prone to crosstalk (but still functional).

Another problem Moon faced is that biological circuits, unlike electronic ones, don't have internal clocks that keep the bits moving through the logic gates in lockstep. If signals progress through layers of gates at different speeds, the output of the entire circuit may be wrong, a problem called a timing fault.

Experiments designed to detect such faults in the synthetic circuit showed that they didn't occur, probably because the chaperones for one layer of logic gates degrades before the transcription factors for the next layer are generated, and this forces a kind of rhythm on the circuit.

Hijacking a bacterium's controller

"We're not trying to build a computer out of biological logic gates," Moon says. "You can't build a computer this way. Instead we're trying to make controllers that will allow us to access all the things biological organisms do in simple, programmable ways."

"I see the cell as a system that consists of a sensor, a controller (the logic circuit), and an actuator," he says. "This paper covers work on the controller, but eventually the controller's output will drive an actuator, something that will do work on the cell's surroundings. "

An synthetic bacterium designed by a friend of Moon's at Nanyang Technological University in Singapore senses signaling molecules released by the pathogen Pseudomonas aeruginosa. When the molecules reach a high enough concentration, the bacterium generates a toxin and a protein that causes it to burst, releasing the toxin, and killing nearby P. aeruginosa.

"Silicon cannot do that," Moon says.

Journal Reference:

1. Tae Seok Moon, Chunbo Lou, Alvin Tamsir, Brynne C. Stanton, Christopher A. Voigt. Genetic programs constructed from layered logic gates in single cells. Nature, 2012; DOI: 10.1038/nature11516

Courtesy: ScienceDaily

New Light Shed On Cancer Risks Associated With Night Work

Night work can increase cancer risk in men, according to a new study published in the American Journal of Epidemiology by a research team from Centre INRS-Institut Armand-Frappier and Centre de recherche du Centre hospitalier de l'Université de Montréal. The study is one of the first in the world to provide evidence among men of a possible association between night work and the risk of prostate, colon, lung, bladder, rectal, and pancreatic cancer and non-Hodgkin's lymphoma.

"Exposure to light at night can lead to a reduced production of the sleep hormone melatonin, inducing physiological changes that may provoke the development of tumours. This hormone, habitually released in the middle of the night in response to absence of light, plays a pivotal role in hormonal functions and in the immune system," explained Professor Marie-Élise Parent of Centre INRS-Institut Armand-Frappier, the study's lead investigator.

Despite finding that night work increases the risk of a number of cancers, the researchers are intrigued by the absence of a relationship between duration of night work and cancer risk found in the study. In theory, an increasing duration in the period of night work would be expected to be accompanied by an increase in the risk of cancer, but the results obtained did not confirm such a tendency. As well as opening up new research avenues, this finding raises questions about the factors that might influence people`s adaptation to night work. Other more targeted research, including Dr. Parent's current research on prostate cancer, will also allow for a more detailed study of the consequences of night work on health.

For this research, Dr. Parent and her team analyzed data from a study on occupational exposure and cancer that was conducted between 1970 and 1985, involving 3,137 men aged 35 to 70 years who had been diagnosed with a cancer at 18 hospitals in the Montreal metropolitan area, compared to a control group of 512 cancer-free individuals from the general population.

The epidemiological study by Marie-Élise Parent, Mariam El-Zein, and Marie-Claude Rousseau of Centre INRS-Institut Armand-Frappier and Javier Pintos and Jack Siemiatycki of Centre de recherche du Centre hospitalier de l'Université de Montréal and Université de Montréal was funded by Health Canada, the National Cancer Institute of Canada , Quebec's Institut de recherche Robert-Sauvé en santé et sécurité au travail, and Fonds de recherche du Québec -- Santé (FRQS).

Journal Reference:

1. M.-E. Parent, M. El-Zein, M.-C. Rousseau, J. Pintos, J. Siemiatycki. Night Work and the Risk of Cancer Among Men. American Journal of Epidemiology, 2012; DOI: 10.1093/aje/kws318

Courtesy: ScienceDaily

 

Early-Earth Cells Modeled to Show How First Life Forms Might Have Packaged RNA

Researchers at Penn State University have developed a chemical model that mimics a possible step in the formation of cellular life on Earth four-billion years ago. Using large "macromolecules" called polymers, the scientists created primitive cell-like structures that they infused with RNA -- the genetic coding material that is thought to precede the appearance of DNA on Earth -- and demonstrated how the molecules would react chemically under conditions that might have been present on the early Earth.

 

The journal Nature Chemistry is posting the research as an Advance Online Publication on 14 October 2012.

In modern biology, all life, with the exception of some viruses, uses DNA as its genetic storage mechanism. According to the "RNA-world" hypothesis, RNA appeared on Earth first, serving as both the genetic-storage material and the functional molecules for catalyzing chemical reactions, then DNA and proteins evolved much later. Unlike DNA, RNA can adopt many different molecular conformations and so it is functionally interactive on the molecular level. In the soon-to-be-published research paper, two professors of chemistry, Christine Keating and Philip Bevilacqua, and two graduate students, Christopher Strulson and Rosalynn Molden, probe one of the nagging mysteries of the RNA-world hypothesis.

"A missing piece of the RNA-world puzzle is compartmentalization," Bevilacqua said. "It's not enough to have the necessary molecules that make up RNA floating around; they need to be compartmentalized and they need to stay together without diffusing away. This packaging needs to happen in a small-enough space -- something analogous to a modern cell -- because a simple fact of chemistry is that molecules need to find each other for a chemical reaction to occur."

To test how early cell-like structures could have formed and acted to compartmentalize RNA molecules even in the absence of lipid-like molecules that make up modern cellular membranes, Strulson and Molden generated simple, non-living model "cells" in the laboratory. "Our team prepared compartments using solutions of two polymers called polyethylene glycol (PEG) and dextran," Keating explained. "These solutions form distinct polymer-rich aqueous compartments, into which molecules like RNA can become locally concentrated."

The team members found that, once the RNA was packed into the dextran-rich compartments, the molecules were able to associate physically, resulting in chemical reactions. "Interestingly, the more densely the RNA was packed, the more quickly the reactions occurred," Bevilacqua explained. "We noted an increase in the rate of chemical reactions of up to about 70-fold. Most importantly, we showed that for RNA to 'do something' -- to react chemically -- it has to be compartmentalized tightly into something like a cell. Our experiments with aqueous two-phase systems (ATPS) have shown that some compartmentalization mechanism may have provided catalysis in an early-Earth environment."

Keating added that, although the team members do not suggest that PEG and dextran were the specific polymers present on the early Earth, they provide a clue to a plausible route to compartmentalization -- phase separation. "Phase separation occurs when different types of polymers are present in solution at relatively high concentrations. Instead of mixing, the sample separates to form two distinct liquids, similar to how oil and water separate." Keating explained. "The aqueous-phase compartments we manufactured using dextran and PEG can drive biochemical reactions by increasing local reactant concentrations. So, it's possible that some other sorts of polymers might have been the molecules that drove compartmentalization on the early Earth." Strulson added that, "In addition to the RNA-world hypothesis, these results may be relevant to RNA localization and function in non-membrane compartments in modern biology."

The team members also found that the longer the string of RNA, the more densely it would be packed into the dextran compartment of the ATPS, while the shorter strings tended to be left out. "We hypothesize that this research result might indicate some kind of primitive sorting method," Bevilacqua said. "As RNA gets shorter, it tends to have less enzyme activity. So, in an early-Earth system similar to our dextran-PEG model system, the full-length, functional RNA would have been sorted and concentrated into one phase, while the shorter RNA that is not only less functional, but also threatens to inhibit important chemical reactions, would not have been included."

The scientists hope to continue their investigations by testing their model-cell method with other polymers. Keating added, "We are interested in looking at compartmentalization in polymer systems that are more closely related to those that may have been present on the early Earth, and also those that may be present in contemporary biological cells, where RNA compartmentalization remains important for a wide range of cellular processes."

This research was funded by the National Science Foundation (grant CHE-0750196).

Journal Reference:

1. Christopher A. Strulson, Rosalynn C. Molden, Christine D. Keating, Philip C. Bevilacqua. RNA catalysis through compartmentalization. Nature Chemistry, 2012; DOI: 10.1038/nchem.1466C

Courtesy: ScienceDaily

Genetic Variants' Role in Increasing Parkinson's Disease Risk Investigated

Boston University School of Medicine (BUSM) investigators have led the first genome-wide evaluation of genetic variants associated with Parkinson's disease (PD). The study, which is published online in PLOS ONE, points to the involvement of specific genes and alterations in their expression as influencing the risk for developing PD.

Jeanne Latourelle, DSc, assistant professor of neurology at BUSM, served as the study's lead author and Richard H. Myers, PhD, professor of neurology at BUSM, served as the study's principal investigator and senior author.

A recent paper by the PD Genome Wide Association Study Consortium (PDGC) confirmed that an increased risk for PD was seen in individuals with genetic variants in or near the genes SNCA, MAPT, GAK/DGKQ, HLA and RIT2, but the mechanism behind the increased risk was not determined.

"One possible effect of the variants would be to change the manner in which a gene is expressed in the brains, leading to increased risk of PD," said Latourelle.

To investigate the theory, the researchers examined the relationship between PD-associated genetic variants and levels of gene expression in brain samples from the frontal cortex of 26 samples with known PD and 24 neurologically healthy control samples. Gene expression was determined using a microarray that screened effects of genetic variants on the expression of genes located very close to the variant, called cis-effects, and genes that are far from the variant, such as those on a completely different chromosome, called trans-effects.

An analysis of the cis-effects showed that several genetic variants in the MAPT region showed a significant association to the expression of multiple nearby genes, including gene LOC644246, the duplicated genes LRRC37A and LRRC37A2 and the gene DCAKD. Significant cis-effects were also observed between variants in the HLA region on chromosome 6 and two nearby genes HLA-DQA1 and HLA-DQA1. An examination of trans-effects revealed 23 DNA sequence variations that reached statistical significance involving variants from the SNCA, MAPT and RIT2 genes.

"The identification of the specific altered genes in PD opens opportunities to further study them in model organisms or cell lines with the goal of identifying drugs which may rectify the defects as treatment for PD," said Myers.

Journal Reference:

1. Jeanne C. Latourelle, Alexandra Dumitriu, Tiffany C. Hadzi, Thomas G. Beach, Richard H. Myers. Evaluation of Parkinson Disease Risk Variants as Expression-QTLs. PLoS ONE, 2012; 7 (10): e46199 DOI: 10.1371/journal.pone.0046199

Courtesy: ScienceDaily

HIV Drug Shows Efficacy in Treating Mouse Models of HER2+ Breast Cancer, Study Suggests

The HIV protease inhibitor, Nelfinavir, can be used to treat HER2-positive breast cancer in the same capacity and dosage regimen that it is used to treat HIV, according to a study published October 5 in the Journal of the National Cancer Institute.

Breast cancer is one of the most common causes of cancer deaths in the U.S. with approximately 39,520 women succumbing to the disease in 2011. HER2-postive breast cancer is known to be more aggressive and less responsive to treatments compared to other types of breast cancer. Nelfinavir has been shown to inhibit the growth of some types of cancers and has been used in clinical trials as either a chemotherapeutic agent or a radiosensitizer for cancer therapy. However, its effect on HER2-positive breast cancer is unknown.

In order to determine the effects of Nelfinavir on HER2-positive breast cancer, Joong Sup Shim, Ph.D., of the Department of Pharmacology and Molecular Sciences at Johns Hopkins School of Medicine and colleagues screened the Johns Hopkins Drug Library and identified a number of inhibitors of breast cancer cells, a subset of which was then used to pharmacologically profile seven genotypically individual breast cancer cell lines. After identifying Nelfinavir as a selective inhibitor of HER2-positive cells, the researchers determined the antitumor activity of the inhibitor in mouse models of human breast cancer.

The researchers found that Nelfinavir inhibited the growth of HER2-positive tumors in mice. They also found that the concentrations of Nelfinavir needed to inhibit HER2-positive cancer cells in vitro are consistent with dosage regimens used for HIV patients. "With a relatively low toxicity profile and much available information on its drug-drug interactions and on pharmacokinetics, Nelfinavir is ready for clinical testing in HER2 breast cancer patients," the authors write, adding that this discovery has, "important implications in the development of Nelfinavir and its analogs as new anticancer agents."

Journal Reference:

1. Joong Sup Shim, Rajini Rao, Kristin Beebe, Len Neckers, Inkyu Han, Rita Nahta, and Jun O. Liu. Selective Inhibition of HER2-Positive Breast Cancer Cells by the HIV Protease Inhibitor Nelfinavir. J Natl Cancer Inst, October 5, 2012 DOI: 10.1093/jnci/djs396

Courtesy: ScienceDaily

A Molecular Scissor Related to Alzheimer’s Disease

An international research team led by the Spanish National Research Council (CSIC) and researchers from Kiel University revealed the atomic‐level structure of the human peptidase enzyme meprin β (beta). The enzyme is related to inflammation, cancer and Alzheimer's Disease and is involved in cellular proliferation and differentiation. The knowledge of the enzyme structure will allow for the development of a new medication type different from those known up to now.

The study was published in the current issue of the Proceedings of the National Academy of Sciences.

"Now that we know how meprin β looks, how it works and how it relates to diseases, we can search for substances that stop its enzyme activities when they become harmful," explains Xavier Gomis-Rüth, researcher at the Molecular Biology Institute of Barcelona, who led the project. Meprin β is an enzyme that is anchored in the outer wall of cells. Its normal function in the human metabolism is to cut off certain proteins, e.g. growth factors, that are also anchored in the cell wall. In this way meprin β releases protein fragments into the environment surrounding the cells -- a natural and normal process, as long as it occurs at a certain intensity. However, under specific circumstances, meprin β may function abnormally, and, for example, releases too many protein fragments. The protein pieces than overdo their natural task in the cell surroundings, causing disorder in the human body. Such disorder typically occurs when inflammation, cancer or Alzheimer's Disease get started.

In their study, the scientists found out that meprin β consists of two identical molecules building a dimeric structure with a cleft in the middle. "We also discovered that the active site cleft is something like the scissor of the enzyme, the actual place where the proteins are cleaved," explains Christoph Becker-Pauly, researcher at the Institute of Biochemistry at Kiel University, and principle investigator of the Kiel Collaborative Research Center 877 „Proteolysis as a Regulatory Event in Pathophysiology." Molecular biologist Gomes-Rüth points out to the next research goal: "We now need to find a substance that fits right into the cleft and will thus block the cleaving activity of meprin β." Such a substance could be the key to new therapeutical drugs against inflammation, cancer or Alzheimer's Disease.

The research has been carried out in collaboration with scientists from Max Planck Institute for Biochemistry and Johannes Gutenberg University Mainz (Germany) as well as University of Bern (Switzerland).

Journal Reference:

1. J. L. Arolas, C. Broder, T. Jefferson, T. Guevara, E. E. Sterchi, W. Bode, W. Stocker, C. Becker-Pauly, F. X. Gomis-Ruth. Structural basis for the sheddase function of human meprin   metalloproteinase at the plasma membrane. Proceedings of the National Academy of Sciences, 2012; 109 (40): 16131 DOI: 10.1073/pnas.1211076109

Courtesy: ScienceDaily