Sunday, July 11, 2010
Mitochondrial Research
2) Evolutionary Systems Biology, Marine Metagenomics, and Paleogenetics.
• The development of mouse models of mitochondrial dysfunction in diabetes and autism.
• The crossroads of mitochondrial function in innate immunity, autoimmunity, aging, stem cell biology, and regeneration.
• The detailed mechanisms of tissue-specific mtDNA replication, damage, copy number control, and extracellular nucleotide signaling via P2X and P2Y receptors in the control of CNV formation and DNA structural diversity during development.
• Develop novel methods for the study of secreted cellular exosomes and their genomics, proteomics, lipidomics, and metabolomics as non-invasive biomarkers of health and disease.
• Developing new methods of environmental DNA isolation and next-generation sequencing to create molecular census tables and trophic architectural maps of contemporary and ancient ecosystems.
Definitions:
Mitochondria: A membrane-enclosed organelle found in eukaryotic cells with 0.5-10 μm diameter. They generate most of the cell’s supply of ATP, they are also involved in signaling, cell differentiation, cell death, cell cycle and cell growth.
Evolutionary systems biology: Quantitative analysis of biological systems
Metagenomics: The study of genetic material recovered directly from environmental samples rather than cultivated microbial clonal cultures.
Paleogenetics: The application of genetics and paleontology “the study of pre-historic life including the organism’s evolution and interaction with the environment, explaining causes rather than observing the experiment’s effects.”
Autism: Neural development disorder defined by impaired social interaction and communication by restricted and repetitive behavior.
P2X receptors : Cation-permeable ion channels in response to ATP.
CNV : Copy number variant, a segment of DNA where the copy-number differences are found by comparison of genomes.
Exosome: A multi-protein complex capable of degrading RNA.
The trophic level of an organism is the position it occupies on the food chain.
Mitochondrial disease symptoms:
• 3 or more organs are affected
• A relapsing disease with measurable slow deterioration
• Unexplained seizures, low blood counts, spasms, blindness, deafness, dementia, atamxia, cerebral palsy, heart failure and muscle weakness.
Inside Mirochondria:
• 3000 proteins. <1% “13 proteins” are encoded by mitochondrial DNA, while >99% are encoded by nucleus DNA. Each cell encodes 10K-15K proteins and mitochondrial proteins are 20-30% of it. Those 13 proteins are involved in electron transport to make ATP.
• 5 copies of ring-shaped DNA encoding 13 mitochondrial proteins. Mitochondrial DNA is the only DNA with maternal inheritance.
Friday, July 9, 2010
Saturday, July 3, 2010
Synthetic Biology Benner and Sismour
My Summary:
There are two types of synthetic biologists. The first group uses unnatural molecules to mimic natural molecules with the goal of creating artificial life. The second group uses natural molecules and assembles them into a system that acts unnaturally. In general, the goal is to solve problems that are not easily understood through analysis and observation alone and it is only achieved by the manifestation of new models. So far, synthetic biology has produced diagnostic tools for diseases such as HIV and hepatitis viruses as well as devices from biomolecular parts with interesting functions. The term “synthetic biology” was first used on genetically engineered bacteria that were created with recombinant DNA technology which was synonymous with bioengineering. Later the term “synthetic biology” was used as a mean to redesign life which is an extension of biomimetic chemistry, where organic synthesis is used to generate artificial molecules that mimic natural molecules such as enzymes. Synthetic biologists are trying to assemble unnatural components to support Darwinian evolution. Recently, the engineering community is seeking to extract components from the biological systems to test and confirm them as building units to be reassembled in a way that can mimic the living nature. In the engineering aspect of synthetic biology, the suitable parts are the ones that can contribute independently to the whole system so that the behavior of an assembly can be predicted. DNA consists of double-stranded anti-parallel strands each having four various nucleotides assembled from bases, sugars and phosphates which are made of carbon, nitrogen, oxygen, hydrogen and phosphorus atoms. In the Watson-Crick model, A pairs with T and G pairs with C although occasionally some diversity exists. This simplification doesn’t exist in proteins. With analysis and observation alone, scientists convince themselves that the paradigms are the truth and if the data contradicts the theory, the data normally is discarded as an error, where synthesis encourages scientists to cross into the new land and define new theories. The same synthesis has long been used in chemistry such as chromatography. The combination of chemistry, biology and engineering can therefore create artificial Darwinian systems.
Friday, July 2, 2010
Dr. Leonidas Bleris Synthetic Biology
Leonidas Bleris, FAS Center for Systems Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138
Components of a living organism, from organs and tissues to single cells and subcellular compartments, exchange and process numerous molecular signals in order to coordinate their activity. When these components fail, they generate characteristic signals that often trigger self-repair processes but can also cause disease when left unchecked. In the not-so-distant future, engineered biomolecular circuits will process information in human cells monitoring in parallel multiple inputs, detecting minute changes, rapidly assessing a patient's condition, and responding in infinitesimal time. Such systems will be used for diagnosing, preventing, treating, and monitoring disease in ways that achieve optimal and highly specific individual health-care, redefining personalized medicine and opening the path to new technologies.
Today, scientists in the cross-sections of disciplines such as biology, chemistry, mathematics, and engineering strive to produce molecular circuits with novel and useful functionalities, in a strikingly similar manner to physicists and engineers that built the fundamental building blocks of computers and modern electronic devices during the 19th century. Similar to a transistor, the basic component of an electrical circuit, with the voltage indicating binary high and low output, in cells a gene can have a binary high and low state depending on the protein concentration. Towards this direction, there are several prototype "biodevices" that operate in cells, such as oscillators, toggle switches, and circuits implementing basic Boolean operations (i.e. AND, OR, NOT logic gates). These devices comprise of genetic and biochemical components such as RNA, DNA fragments, proteins, and inducer molecules.
We have constructed a de novo molecular information-processing gene network that operates in human kidney cells. This molecular circuit is based on RNA interference (RNAi), a mechanism for RNA-guided regulation of gene expression. We show that the RNAi pathway in human cells can form a molecular computing module capable of evaluating arbitrary Boolean expressions on endogenous cues. We experimentally demonstrate in human kidney cells the direct evaluation using exemplary expressions in standard forms with up to five logic variables.
Design and Optimization of An RNAi Based Biomolecular Logic Circuit
Today, scientists in the cross-sections of disciplines such as biology, chemistry, mathematics, and engineering strive to produce molecular circuits with novel and useful functionalities, in a strikingly similar manner to physicists and engineers that built the fundamental building blocks of computers and modern electronic devices during the 19th century. Similar to a transistor, the basic component of an electrical circuit, with the voltage indicating binary high and low output, in cells a gene can have a binary high and low state depending on the protein concentration. Towards this direction, there are several prototype "biodevices" [1-5] that operate in cells, such as oscillators, toggle switches, and circuits implementing basic Boolean operations (i.e. AND, OR, NOT logic gates). These devices comprise of genetic and biochemical components such as RNA, DNA fragments, proteins, and inducer molecules.
In [6] we presented an experimental implementation of modules that can evaluate logic expressions in human kidney cells, using disjunctive and conjunctive normal forms (DNF and CNF). Since any Boolean expression can be represented in CNF and in DNF form these modules allow for the evaluation of any arbitrary logic or condition in vivo. The CNF and DNF modules use a combination of transcriptional and post-transcriptional regulation pathways as the underlying molecular "hardware". The logic expressions are encoded in a multigene network as the "software", and the inputs, i.e. the truth values of the variables, are encoded by the presence or absence of small interfering RNAs (siRNAs) utilizing the RNA interference pathway. The result of the evaluation is read out using a fluorescent reporter protein. We conducted experiments to prove the feasibility of the computation framework using transient cotransfections of the software segment genes and siRNA molecules. We present experimental results of subsequent generations of the evaluator, starting from two-input logical AND and OR operations to more complex five variable cases. We highlight the approach used for the optimization of the performance of this circuit and discuss extensions and limitations.
[1]. Weiss, R., Homsy, G.E., Knight, T.F. Toward in vivo digital circuits. DIMACS workshop on evolution in computation (1999)
[2]. Benenson, Y., Gil, B., Ben-Dor, U., Adar, R. & Shapiro, E. An autonomous molecular computer for logical control of gene expression. Nature 429, 423-429 (2004)
[3]. Stojanovic, M. N., Stefanovic, D. A deoxyribozyme-based molecular automaton. Nature Biotechnology 21, 1069-1074 (2003)
[4]. Hasty J, McMillen D and Collins JJ. Engineered gene circuits. Nature 420: 224-230 (2002).
[5]. Elowitz MB, Leibler S. A Synthetic Oscillatory Network of Transcriptional Regulators. Nature 403, 335-8 (2000)
[6]. K. Rinaudo*, L. G. Bleris*, R. Maddamsetti, S. Subramanian, R. Weiss, Y. Benenson. A Universal RNAi-based Logic Evaluator that Operates in Mammalian Cells. Nature Biotechnology 25, 795-801 (2007) (* Equal contribution)
A universal RNAi-based logic evaluator that operates in mammalian cells.
Rinaudo K, Bleris L, Maddamsetti R, Subramanian S, Weiss R, Benenson Y.
Abstract
Molecular automata that combine sensing, computation and actuation enable programmable manipulation of biological systems. We use RNA interference (RNAi) in human kidney cells to construct a molecular computing core that implements general Boolean logic to make decisions based on endogenous molecular inputs. The state of an endogenous input is encoded by the presence or absence of 'mediator' small interfering RNAs (siRNAs). The encoding rules, combined with a specific arrangement of the siRNA targets in a synthetic gene network, allow direct evaluation of any Boolean expression in standard forms using siRNAs and indirect evaluation using endogenous inputs. We demonstrate direct evaluation of expressions with up to five logic variables. Implementation of the encoding rules through sensory up- and down-regulatory links between the inputs and siRNA mediators will allow arbitrary Boolean decision-making using these inputs.
