69 posts · 27,928 views
To master molecular mechanisms we need to know how they works within the whole organism. My vision is that new developments in genetic engineering will branch new applications for reporter genes, not necessarily confined to report transcriptional regulation. In my blog I trend advances in such a 'reportergenomics', a discipline at the crossroad between synthetic and system biology.
96well
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- June 28, 2010
- 12:26 PM
- 90 views
Photoactivation through the central dogma of molecular biology
by 96well in Reportergene
Photoactivation is the property of a molecule of being capable of pronounced changes in its chemical properties in response to irradiation with light of a specific wavelength and intensity. This feature provides unique possibilities for the design of new strategies aimed at the spatio-temporal deciphering of molecular pathwhays occuring in living cells, organelles and intracellular molecules. Here, I spotlight two recent applications: from photoactivable nucleotides to photoactivable proteins.
RNA-binding motifs
UV-light irradiation has been used in the past to isolate RNAs bound to specific binding proteins (RBPs) but the discovery rate was inefficient because of problems in separating signal from background. Haffner and colleagues exploited photoactivable nucleosides as efficient and non-toxic crosslinkers that are well incorporated into the nascent RNA. Cells are grown in the presence of X mM of 4-thiouridine (4SU): 4SU-substituted RNA is 'activated' by UV light at 365 nm and specifically crosslinks to its RNA-binding partner proteins. Interestingly, if crosslinked and de-crosslinked (after immunoprecipitation of the RNA-bound-protein) 4SU is then mis-paired with guanine (G) instead of adenine (A) during the reverse transcription to cDNA. This allows the precise mapping of the binding sites by scoring the G/A transitions in the cDNA sequence. The researchers used this technique to define the binding motifs of 13 RBPs. (Haffner et al., Cell 2010)
Proteasome dynamics
Fluorescent reporters have already been used in the past to monitor proteosomal degradation, but effects on reporter degradation are difficult to separate from effects on reporter synthesis. Hamer and colleagues designed a reporter in which these processes can be easily distinguished. The green-to-red photoconvertible protein Dendra2 is fused to a form of ubiquitin that targets Dendra2 for proteosomal degradation. By observing only the photoconverted red form of the reporer, effects on its synthesis can be ruled out. The researchers used this reporter to monitor the cell type- and age-dependence of ubiquitin proteosome activity in living C.elegans. (Hamer et al., Nature Methods 2010)
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Hafner, M., Landthaler, M., Burger, L., Khorshid, M., Hausser, J., Berninger, P., Rothballer, A., Ascano Jr., M., Jungkamp, A., & Munschauer, M. (2010). Transcriptome-wide Identification of RNA-Binding Protein and MicroRNA Target Sites by PAR-CLIP Cell, 141 (1), 129-141 DOI: 10.1016/j.cell.2010.03.009
Hamer, G., Matilainen, O., & Holmberg, C. (2010). A photoconvertible reporter of the ubiquitin-proteasome system in vivo Nature Methods, 7 (6), 473-478 DOI: 10.1038/nmeth.1460
... Read more »
Hafner, M., Landthaler, M., Burger, L., Khorshid, M., Hausser, J., Berninger, P., Rothballer, A., Ascano Jr., M., Jungkamp, A., & Munschauer, M. (2010) Transcriptome-wide Identification of RNA-Binding Protein and MicroRNA Target Sites by PAR-CLIP. Cell, 141(1), 129-141. DOI: 10.1016/j.cell.2010.03.009
- April 29, 2010
- 12:34 PM
- 127 views
non-natural reporter proteins unleashed to the red
by 96well in Reportergene
After the pioneering works of Schultz's group and Chamberlin's group in 1989, bio-synthetic incorporation of non-natural amino-acids into proteins has been largely explored in chemical biology. Direct evolution of new tRNAs able to carry 'new' amino-acids, combined with codon extension to the 4th base has provided us with the ability to expand the chemical functionality of proteins by introducing new chemical moieties into their backbone. More importantly, we can do this in a genetically-heritable fashion. This new variety of protein opens several avenues for synthetic biology. Here, I spotlight two new non-natural reporter proteins.
a reporter for infrared spectroscopy
The non-natural amino-acid p-azido-l-phenylalanine (azF) vibrates in a window which is free from other protein vibration, this feature has been exploited to create a new infrared protein probe by incorporating azF into a G-coupled Protein Receptor (Rhodopsin). This allow to study GPCR's conformational changes by Fourier-transform infrared (FTIR) analysis. Upon the receptor activation, early conformational changes were observed.
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Ye, S., Zaitseva, E., Caltabiano, G., Schertler, G., Sakmar, T., Deupi, X., & Vogel, R. (2010). Tracking G-protein-coupled receptor activation using genetically encoded infrared probes Nature, 464 (7293), 1386-1389 DOI: 10.1038/nature08948
non-natural red bioluminescence
The non-natural amino-acid 4-methoxy-phenylalanine has been incorporated into the position F82 of the reporter aequorin, resulting in a significant (44 nm) red-shift of the aequorin bioluminescence.
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Rowe, L., Ensor, M., Mehl, R., & Daunert, S. (2010). Modulating the Bioluminescence Emission of Photoproteins by Site-Directed Incorporation of Non-Natural Amino Acids
ACS Chemical Biology DOI: 10.1021/cb9002909
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Ye, S., Zaitseva, E., Caltabiano, G., Schertler, G., Sakmar, T., Deupi, X., & Vogel, R. (2010) Tracking G-protein-coupled receptor activation using genetically encoded infrared probes. Nature, 464(7293), 1386-1389. DOI: 10.1038/nature08948
Rowe, L., Ensor, M., Mehl, R., & Daunert, S. (2010) Modulating the Bioluminescence Emission of Photoproteins by Site-Directed Incorporation of Non-Natural Amino Acids . ACS Chemical Biology, 2147483647. DOI: 10.1021/cb9002909
- March 30, 2010
- 03:52 PM
- 157 views
toward a better drug classification
by 96well in Reportergene
Just a bit of self-promotion about my last paper. Despite the superiority of longitudinal vs cross-sectional studies, the dynamics of drug action are poorly explored in pre-clinical studies. Little is known about how drugs affect the activity of their intended target over time. Here, we used a longitudinal imaging approach to accurately follow the state of transcriptional activity of one drug target (the estrogen receptor) in 8 anatomical areas of living ERE-luc reporter mice over 21 consecutive days of hormone replacement therapy with 10 different Selective Estrogen Receptor Modulators (SERMs).
This is a 2006: I checked the accuracy of the algorithm measuring the area under the curve by weighing small pieces of paper. My bench-mates are still laughing...We found that each SERM caused tissue-specific oscillations on ER transcriptional activity which were predictive of the drug structure (after rational extraction of meaningful molecular descriptors).
For more than one century, the measure of drug structure-activity relationships has been based on mathematical equations describing the interaction of the drug with its biological receptor. This is now obsolete, as 'binding' does not necessarily spell 'function'. Here, a systematic study of spatio-temporal effects is proposed as a measure of drug efficacy for the classification of pharmacologically active compounds.We conclude that the use of reporter mouse facilitates the identification of SERMs able to mimic the physiological estrous cycle and anticipates the possibility of a reverse approach in medicinal chemistry where the space-temporal plot of target activity drives the identification of subtle structure-activity relationships.
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Rando, G., Horner, D., Biserni, A., Ramachandran, B., Caruso, D., Ciana, P., Komm, B., & Maggi, A. (2010). An Innovative Method to Classify SERMs Based on the Dynamics of Estrogen Receptor Transcriptional Activity in Living Animals Molecular Endocrinology, 24 (4), 735-744 DOI: 10.1210/me.2009-0514
Rando G, Biserni A, Ciana P, & Maggi A (2010). Profiling of drug action using reporter mice and molecular imaging. Methods in molecular biology (Clifton, N.J.), 602, 79-92 PMID: 20012393
Rando G, Arca S, Casiraghi E, Campadelli P & Maggi A (2009). Automatic Segmentation of Mouse Images. Proc 10th European Congress of International Society for Stereology, Bologna, Italy, 2009 link: abstract
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Rando, G., Horner, D., Biserni, A., Ramachandran, B., Caruso, D., Ciana, P., Komm, B., & Maggi, A. (2010) An Innovative Method to Classify SERMs Based on the Dynamics of Estrogen Receptor Transcriptional Activity in Living Animals. Molecular Endocrinology, 24(4), 735-744. DOI: 10.1210/me.2009-0514
- March 2, 2010
- 06:01 PM
- 169 views
A darwinian legacy OR Why we need fluorescent rabbits
by 96well in Reportergene
My post about fluorescent rabbits is gaining a momentum on the Flickr group 'Bunny Lovers Unite' and in the Rabbitmatch's blog. Most people ask itself: WHY making fluorescent bunnies? And others feel outraged.
Animal research is long debated, and my hope is that the development of new reporter probes would allow to reconsider current research protocols while increasing the scientific significance of the experiments done, this is the focus of my current research. Here, a take opportunity of this little exposure to the non-science world to say why WE CAN make better animal research.
From a modern mechanical and operational perspective, life relies on the interaction between a sworm of molecules and their hierarchical organization into structures in which we distinguish a functional unity (i.e., organelles, cells, tissues, organs and systems). It is current hope and belief that a whole, systematic decoding of such minute, living, interacting building blocks that are bio-molecules, will grant a better comprehension of life mechanisms. From more than one century, we learn how to identify and discriminate those molecules, by means of iterative, multidimensional separation techniques (i.e., 2D electrophoresis, tandem mass spectrometry). This provided us with the awareness of the vast compilation of molecules and their distribution among different single cells and, ultimately, into the whole biome.
Laboratory animals were the main source of specimens to put under our microscopes. However, from the slides, we progressively realized that the picture we were painting was actually a static still-life, without any motion. This is not surprising, given the fact that most of the research conducted so far have been based on post-mortem analyses. Worst, such frozen picture renders difficult to discriminate causes from consequences. Animal sacrifice raises scientific, ethical and economical concerns, moreover the paradox of studying life from dead samples, may reasonably induce to wonder about the artefactual side of the knowledge generated so far. Not only terminal euthanasia, but each animal distress poses the same doubts: are we acquiring a distressed knowledge?
Before the molecular age, giants like Darwin and Mendel revolutionized life sciences with a systematic observation of animal and vegetal living specimen in their natural environment (the galapagos) with minor to null human intervention other than observation. The point is: shall we systematically observe (and understand) molecular life into a living, awake, freely moving animal in its natural environment? Can we imagine a day in which our preferred pet, in the security of our home, would allow to make scientific discoveries because he/she possess a radio-gene able to communicate its physiological and molecular status to our wireless router which will promptly share this information to the scientific community online?
I'm dreaming about normal happy pets, that can be turned into research contributors just through a simple injection. This is science-fiction but we aren't very far from such a goal. Recent advances in molecular imaging led us to dream a clean future for animal research. Take the simplest molecule: H2O. Its diamagnetism led clinicians to monitor water in space and time by means of magnetic resonance imaging (the same can be done for fat). In 2003, we were able to monitor something more elegant than the space-temporal profile of a single molecule in a healthy living mouse: by means of optical bioluminescence imaging, my PhD mentor was able to observe in cycling female mice, not a molecule, but its molecular activity (specifically, the activity of the estrogen receptor, a hormone-regulated transcription factor).
This goal, was achieved by the conceptualization of so-called reporter mice: models engineered in order to allow the external non-invasive monitoring of any selected molecular mechanism in the full respect of animal’s dignity. This is, in other words, the ability to observe the molecular life into a living whole organism. A Darwinian legacy.
The most interesting feature of longitudinal imaging with living animal systems, however, is its excellent potential to Reduce the number of animals because animal sacrifice is NOT needed and each animal is its own control. We need now to improve this technology (toward the radio-gene) to Refine current methods by providing the opportunity to study molecular circuits systematically in 4 dimensions, furthermore new technology will abolish the pain for the animals (and voluntary humans). What we need now, are engineers providing multiplexing abilities and better resolutions (in space and in time). Molecular imaging should be considered a very valid Replacement alternative, and we need to shift mathematical engineers and bioinformaticians from still-life omics data set, to living data set. We need to hope and belief to alternative ways to conduct animal experimentation. Animals aren't humans. We need to develop safer, safest technologies to study gene networks in animals in order to validate the safety and to eventually apply this 'radio-genes' to humans, to ourselves. Nosce te ipsum (know thyself, said Socrates). This blog tales recent advances toward this vision, the vision of a clean, efficient, innocent biomedical research.
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Ciana, P., Raviscioni, M., Mussi, P., Vegeto, E., Que, I., Parker, M., Lowik, C., & Maggi, A. (2003). In vivo imaging of transcriptionally active estrogen receptors Nature Medicine, 9 (1), 82-86 DOI: 10.1038/nm809
Maggi A, & Rando G (2009). Reporter mice for the study of intracellular receptor activity. Methods in molecular biology (Clifton, N.J.), 590, 307-16 PMID: 19763513
Rando G, Biserni A, Ciana P, & Maggi A (2010). Profiling of drug action using reporter mice and molecular imaging. Methods in molecular biology (Clifton, N.J.), 602, 79-92 PMID: 20012393
... Read more »
Ciana, P., Raviscioni, M., Mussi, P., Vegeto, E., Que, I., Parker, M., Lowik, C., & Maggi, A. (2002) In vivo imaging of transcriptionally active estrogen receptors. Nature Medicine, 9(1), 82-86. DOI: 10.1038/nm809
Maggi A, & Rando G. (2009) Reporter mice for the study of intracellular receptor activity. Methods in molecular biology (Clifton, N.J.), 307-16. PMID: 19763513
- February 17, 2010
- 01:11 PM
- 194 views
Molecular surgery: playing with network edges
by 96well in Reportergene
Protein X interacts with protein Y, what are the phenotypic consequences? And what is the impact of the X-Y partnership in the whole protein-protein interaction network? To address this question, scientists often remove specific network nodes by eliminating (knock-out) or downregulating (knock-down) the gene encoding one protein product (i.e. X). This is a poor strategy, because usually X interacts not only with Y, but also with P, Q, R, S, T, U, V, W and Z. Thus, X-KO strategy is too much invasive and prone to promptly activate compensatory mechanisms in the newtork redundancy program, making the phenotypic interpretation a pain in the ASS. To better understand networks, one should be less invasive and knock-down edges other than nodes.
This new 'molecular edge surgery' can be done now, thanks to a reporter gene: the yeast Cytosine Deaminase (yCD) and to Po Hien Ear and Stephen Michnick and their Nature Methods paper. The yCD reporter can be succesfully splitted and used in classic protein-fragment complementation assays (PCA). The measure of reporter activity in this case is life or death, as yCD allows both positive and negative selection in yeast. So, if protein X interacts both with Y and Z, the molecular edge surgery is done performing first the selection with a library of mutant alleles X* screened against Z (selected for death: disruption of X-Z interaction), the same mutants are then screened against Y (selected for growth: retaining X-Y interaction).
At the end of the streamlined selection one should obtain 'edgetic' mutants preserving the X-Y edge but eliminating the X-Z edge of the protein-protein interaction network. The yCD-PCA assay should provide more granular insights into the relationship between loss of a specific edge on a network and the Phenotype and eventually trace those consequences back to the protein structure. This seems also a beautiful technique for prototyping drugs able to antagonize one 'bad' protein-protein interaction, while maintaining the 'good' one.
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Ear, P., & Michnick, S. (2009). A general life-death selection strategy for dissecting protein functions Nature Methods, 6 (11), 813-816 DOI: 10.1038/NMETH.1389
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Ear, P., & Michnick, S. (2009) A general life-death selection strategy for dissecting protein functions. Nature Methods, 6(11), 813-816. DOI: 10.1038/NMETH.1389
- February 11, 2010
- 01:43 PM
- 188 views
Are you a motivated labmate?
by 96well in Reportergene
I went through this little gift from Uri Alon: its essay appeared in Molecular Cell which aims to conjugate psychological principles to the every-day lab routine for improving motivation. How Uri Alon improves the motivation of his lab? He try to balance three fundamental needs of any scientist: competence, autonomy and social connectedness, for instance:I make our weekly group meeting an event that enhances social connectedness. The first half hour of the two hour meeting is devoted to nonscience. This at first may seem to eliminate one quarter of the time for talking science, but in the long term, gains from increased motivation more than make up for any losses.I'm a little bit skeptical about reading psychologicalities to increase self-motivation and productivity, as I think that to get things done the best is stopping reading this and making something concrete. However, I was in my bed after a long productive day and Molecular Cell is Molecular Cell period. The essay is also well-written. The author does not explain how to recognize symptoms of non-motivation, so I add here my personal thermometer: the measure of my degree of motivation is the laziness taken to duly complete my lab-book.
Post scriptum:
research blogging is a way to increase social connectedness in my case, but after a while I realized that also my competences were increased: I remember more the studies I blog.
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Alon, U. (2010). How to Build a Motivated Research Group Molecular Cell, 37 (2), 151-152 DOI: 10.1016/j.molcel.2010.01.011
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Alon, U. (2010) How to Build a Motivated Research Group. Molecular Cell, 37(2), 151-152. DOI: 10.1016/j.molcel.2010.01.011
- January 20, 2010
- 02:04 PM
- 209 views
Camel vs Jellyfish: a battle with green fluorescence
by 96well in Reportergene
Does the spectral properties of GFP can be modulated by antibody-derivatives? To explore this hypothesis, Axel Kirchhofer and colleagues from Munich Center for Advanced Photonics have designed a number of NanoBodies (NBs) to bind to GFP. (Nanobodies? They are small, antigen-binding, single-domain polypeptides derived from some camelid antibodies). The authors found NBs could increase or decrease GFP fluorescence: in fact, co-chystallization of GFP-NB complexes revealed NBs inducing subtle changes in the chromophore environment. The camel advantage is that, in contrast to conventional antibodies, small, stable Nanobodies are functional in living cells. Exploting this feature, a beautiful experiment was done: a NB 'Enhancer' able to shift the absorption spectra of GFP was first confined into nucleus by means of a lamin-B1 tag. This transformed it in a ratiometric biosensor for subsequent nuclear traslocation of GFP-'transcription factors'. In conclusion, the NB ability to modify the conformation of proteins was demonstrated in living cells anticipating new opportunities for bioimaging studies of cell signaling.
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Kirchhofer, A., Helma, J., Schmidthals, K., Frauer, C., Cui, S., Karcher, A., Pellis, M., Muyldermans, S., Casas-Delucchi, C., Cardoso, M., Leonhardt, H., Hopfner, K., & Rothbauer, U. (2009). Modulation of protein properties in living cells using nanobodies Nature Structural & Molecular Biology, 17 (1), 133-138 DOI: 10.1038/nsmb.1727... Read more »
Kirchhofer, A., Helma, J., Schmidthals, K., Frauer, C., Cui, S., Karcher, A., Pellis, M., Muyldermans, S., Casas-Delucchi, C., Cardoso, M.... (2009) Modulation of protein properties in living cells using nanobodies. Nature Structural , 17(1), 133-138. DOI: 10.1038/nsmb.1727
- January 19, 2010
- 11:00 PM
- 320 views
Green Fluorescent Rabbit
by 96well in Reportergene
A new study demonstrates the feasibility of using a lentiviral approaches to create transgenic rabbits with more efficiency than classical pronuclear injection transgenesis developed in rabbits two decades ago.
Tracing Ruppy, the reporter-dog, the enhanced transgenic bunny carries a green fluorescent protein. Rabbits are still used as laboratory animals as they are genetically closer to primates and are large enough to allow safe and secure blood sampling compared to mice. Thus, transgenic rabbits are promising models for cardiovascular research.... Read more »
Hiripi, L., Negre, D., Cosset, F., Kvell, K., Czömpöly, T., Baranyi, M., Gócza, E., Hoffmann, O., Bender, B., & Bősze, Z. (2010) Transgenic rabbit production with simian immunodeficiency virus-derived lentiviral vector. Transgenic Research. DOI: 10.1007/s11248-009-9356-y
- January 13, 2010
- 06:10 PM
- 404 views
EXTassays, toward maturity of RNA reporters
by 96well in Reportergene
I read with some interest a recent Nature Methods paper appeared this January. Anna Botvinnik and colleagues from Max Planck Institute, conceived a new reporter system able to measure receptor activation (receptor dimerization), downstream signaling (adapter recruitment) and subsequnent cis-regulatory responsive elements transactivation efficacies by...
...no, you don't need a 64-milion new-generation machine, you need Trizol!
As I reviewed in my first 2010 post, there is a trend to develop multiple reporter into a library ready to transfect. In this paper, the authors just coupled each reporter with a unique expressed oligonucleotide tag, transfected the library, isolated the reporter mRNAs as a pool and analyzed it by hybridization to microarray. In brief, this is EXTassay. Wondering about transfection efficiency? Owing to its small scale, plasmid DNA was not denaturated by Trizol and was localized in the acqueous phase (they said), so it can be isolated and hybridized to serve as calibrator.
The trick to probe receptor dimerization relies in the genetic manipulation of the receptor gene, and it is quite similar to the Tango assay. Other than being solely a receptor, the protein in exam is coupled in frame with partial TEV recombinase and a GV transcription factor. Upon receptor dimerization (activation) follows reconstitution of a TEV protease and cleavage of GV, which goes in the nucleus where it can find a corresponding GV-reporter.
Despite I'm still uncomfortable with the idea to interrogate receptor activity by receptor transfection, I start feeling RNA-reporters being on the road toward maturity, and I admit: probably I'have been too much severe with Tango assays in January 2008. I should think at this when planning my future experiments, there is more other than luciferase in 2010.
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Botvinnik, A., Wichert, S., Fischer, T., & Rossner, M. (2010). Integrated analysis of receptor activation and downstream signaling with EXTassays Nature Methods, 7 (1), 74-80 DOI: 10.1038/nmeth.1407
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Botvinnik, A., Wichert, S., Fischer, T., & Rossner, M. (2010) Integrated analysis of receptor activation and downstream signaling with EXTassays. Nature Methods, 7(1), 74-80. DOI: 10.1038/nmeth.1407
- January 4, 2010
- 12:00 AM
- 271 views
2010: luminometers OR sequencers?
by 96well in Reportergene
Next generation sequencing (solexa, illumina, 454) is offering a new opportunity for the design of multiplexed reporter assays. With the notable 2007 exception of Brainbow (in which however, it was not possible to discriminate the origin of the 90 or more observed fluorescent colors because they come from random recombination), simply the co-detection of more than three fluorescent proteins is very challenging in real life because of spectral overlapping and other shortcomings. Early in 2008, I blogged about a new method to detect more than 30 reporters simultaneously: instead of coding for a protein, each reporter was coding for a very similar mRNA. The difference between each mRNA was just a 5-nt lenght: i.e. reporter mRNA #1 was 100 nt long, mRNA #2 was 105 nt, mRNA #3 was 110 nt and so on. Once transfected, the library of reporters was reverse-transcribed with the same primer pairs and then analyzed and semi-quantitated by capillary electrophoresis. Today, forget electrophoresis, and think parallel sequencing. The same plasmid backbone can be coupled to an oligo barcode! The work of Patwardhan et al. open this way: at Washington, they were bothering about the effect of each possible mutation in a promoter, one base at a time. By means of synthetic saturation mutagenesis, mutant promoters were obtained by parallel solid synthesis and then released into solution, resulting in a complex library. The trick was that each oligo in the library was designed to include a unique barcode sequence downstream of the promoter's transcription start site. One library aliquot was used in an in vitro transcription reaction to make RNA from the promoter (subsequently reversely transcribed). The second aliquot was amplified using PCR only. High-throughput illumina sequencing has been carried out and quantitative comparison between the RNA barcode and the DNA barcode was used to give a measure of promoter effectiveness in a way that seems to me more clean than our classical beta-gal normalization. Ok, at the moment the technology is applied only in vitro, but the concept is clear: next generation sequencing can help the reportergenomic community. Give me a barcode plasmid and I'm going to make the reporter mouse.... Read more »
Patwardhan, R., Lee, C., Litvin, O., Young, D., Pe'er, D., & Shendure, J. (2009) High-resolution analysis of DNA regulatory elements by synthetic saturation mutagenesis. Nature Biotechnology, 27(12), 1173-1175. DOI: 10.1038/nbt.1589
- December 23, 2009
- 08:11 AM
- 393 views
bacteria towing santa's wagon?
by 96well in Reportergene
Bacteria swim in water solution with a random movement resembling brownian motion. Imagine they while impacting randomly on a micro-gear. They will transfer some energy to the gear, but due to random movements the resultant will not provide any directed motion. However, you should remember from physics101 that a principle for the conservation of the force does NOT exist: imagine each tooth of the gear as a lever arm, and do design gears with asymmetric teeth. In this way, bacteria impacting on one tooth side, will transfer more energy than bacteria impacting on the other side. And here comes the magic: motion!
By conventional photolitography, Sokolov and colleagues from Argonne National Laboratory, report on PNAS the design of microscopic gears (6 ug mass) able to extract useful work from swimming Bacillus subtilis sworms. According to their calculations, few hundred bacteria work together in order to turn the gear at a velocity of 1-2 rpm, generating femtowatt power.
Here the concept is that brownian motion can generate directed motion (in non-equilibrium conditions). Since Middle Age, oxen, horses and other animals have been used to generate power to move mills and other machines (in non-equilibrium conditions). Bacteria, and perhaps other floating cells/molecules might provide new energy sources for new micro-machines. Applications are wide and may impact also molecular and synthetic biology. What about designing proteins with asymmetrical teeth? Merry Christmas.
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Sokolov, A., Apodaca, M., Grzybowski, B., & Aranson, I. (2009). Swimming bacteria power microscopic gears Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.0913015107
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Sokolov, A., Apodaca, M., Grzybowski, B., & Aranson, I. (2009) Swimming bacteria power microscopic gears. Proceedings of the National Academy of Sciences. DOI: 10.1073/pnas.0913015107
- November 28, 2009
- 07:50 AM
- 83 views
Sorry, I'm experiencing a temporal drift toward metaphysics
by 96well in Reportergene
As we know it, our molecular life as individuals starts with a fusion between a female oocyte and a male sperm cell. When our mother was born, she got already in her ovary that small not-matured oocyte that than contributed to our first half cell at the time of ovulation several years later. Conversely, it is believed that our father at the time of conception, just donate our second half cell by means of a sperm cell (randomly) produced de novo.
Now, Zhuoru Wu and her colleagues at the University of Texas Southwestern Medical Center in Dallas shows that when stem cells in the rat testes are at developmental crossroad, they have two choices: procrastinate becoming again stem cells or differentiate into sperm. The researchers found that when grown in the same culture medium, stem cells gave rise to more stem cells that sperm. Thus, if we want for a while being fascinated by predestination, it is conceptually possible that both the female oocyte and the male stem-to-sperm cell that initiated our life were waiting each other from the very beginning of our parent's life.... Read more »
Wu, Z., Luby-Phelps, K., Bugde, A., Molyneux, L., Denard, B., Li, W., Suel, G., & Garbers, D. (2009) Capacity for stochastic self-renewal and differentiation in mammalian spermatogonial stem cells. The Journal of Cell Biology, 187(4), 513-524. DOI: 10.1083/jcb.200907047
- November 25, 2009
- 08:27 AM
- 529 views
Malaria in 3D: bioluminescence imaging
by 96well in Reportergene
In a recent Plos One paper, Ploemen and colleagues (Nijmegen Medical Centre) use previously generated luciferase-bearing malaria parasites (PbGFP-Luccon) to study the spatio-temporal development of malaria infection in liver of living infected mice. The final aim of the paper is to propose 3D-imaging to explore the effect of drug and vaccines on P. falciparum infection without surgery and other invasive methodologies in the mouse. Interestingly, they report good tri-dimensional plasmodium tracking in the liver, which can be followed over time.Modified from Ploemen et al., Plos ONE 2009A 3D approximation is obtained through rotational axis bioluminescence imaging through a CaliperLS workstation: eight imaging views from different advantage points are determined. At each angle view, both the picture of the mouse and its luciferase signal at 3 different wavelengths are acquired. Then, the algorithm tries, from surface data, to reconstruct what is happening inside the mouse by providing an approximate 3D-reconstruction. Last, a digital mouse atlas is overlaid onto the 3D diffuse tomography reconstruction to obtain anatomical reference points. The Caliper algorithm is longer to be perfect (see 2008 post on new developments in 3D-bioluminescence) in fact it is based on the assumption that the mouse is optically homogeneous. However, it seems to perform pretty well at least for a parenchimal organ like the liver (see picture). Indeed, also fluorescence tomography works well in liver (see post on infrared fluorescent protein).In conclusion, at least for liver, it is possible to follow a reporter with enhanced spatio-temporal resolution. Since in liver, contrarily to the whole mouse, genetic engineering can be successfully performed with simple hydrodynamic injections, one would expect lot of progress in the future of molecular hepatology.Ploemen, I., Prudêncio, M., Douradinha, B., Ramesar, J., Fonager, J., van Gemert, G., Luty, A., Hermsen, C., Sauerwein, R., Baptista, F., Mota, M., Waters, A., Que, I., Lowik, C., Khan, S., Janse, C., & Franke-Fayard, B. (2009). Visualisation and Quantitative Analysis of the Rodent Malaria Liver Stage by Real Time Imaging PLoS ONE, 4 (11) DOI: 10.1371/journal.pone.0007881
... Read more »
Ploemen, I., Prudêncio, M., Douradinha, B., Ramesar, J., Fonager, J., van Gemert, G., Luty, A., Hermsen, C., Sauerwein, R., Baptista, F.... (2009) Visualisation and Quantitative Analysis of the Rodent Malaria Liver Stage by Real Time Imaging. PLoS ONE, 4(11). DOI: 10.1371/journal.pone.0007881
- October 28, 2009
- 01:35 PM
- 448 views
Chromophores, a new class of reporters
by 96well in Reportergene
A new Nature letter has the potential to abnormally extend (until extinction) the whole spectrum of reporter genes. So far, "reporters" were those genes coding for an easily detectable product (i.e., those coding for fluorescent or luminescent proteins). Wei Min and other Harvard's colleagues introduced a new technique, namely stimulated emission microscopy, that seems able to turn into mini-lasers any non-fluorescent light-absorbing molecule. It means that several chromophores, such as haemoglobin and cytochromes, can now be directly detected through a new contrast mechanism for optical microscopy which is orders of magnitude more sensitive than absorption, is not subject to interference from other chromophores in the sample, and is amenable to three-dimensional sectioning. This open the race to the intelligent design of new chromo-reporters able to produce images of unlabelled, non-fluorescent molecules at sub-diffraction (nanoscale) resolution. Seeing is believing. The potential to kill the field of reporters is easily explained: who ever need a "reporter" when you can spy in a cell the whole bunch of molecular processes with resolutions at googlemeter per googlesecond?Min, W., Lu, S., Chong, S., Roy, R., Holtom, G., & Xie, X. (2009). Imaging chromophores with undetectable fluorescence by stimulated emission microscopy Nature, 461 (7267), 1105-1109 DOI: 10.1038/nature08438
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Min, W., Lu, S., Chong, S., Roy, R., Holtom, G., & Xie, X. (2009) Imaging chromophores with undetectable fluorescence by stimulated emission microscopy. Nature, 461(7267), 1105-1109. DOI: 10.1038/nature08438
- September 11, 2009
- 01:15 PM
- 584 views
engineering photo-activable proteins
by 96well in Reportergene
Plants contain proteins subjected to conformational changes in direct response to light irradiation. Moieties of those proteins, like the LOV2 domain from the Avena sativa Phototropin1 can be used to introduce light-operated switches onto other functional proteins. In a recent letter to Nature, Yi Wu and colleagues (Carolina University) poked at the Stratagene Quickchange kit to obtain a constitutive active Rac protein that was coupled to the vegetable LOV2 light switch using an overlapping PCR approach. The result of such a cut and paste was genetically encoded into HeLa, HEK93 and MEF/3T3 cell lines. Then, by irradiating whole cells or even localized micro-spot on the cell surface, PA-Rac1 was sufficiently photo-activated to generate polarized cell movements. In other words, light was controlling the motility of living cells via photoactivable Rac. Structural studies indicate that the Rac-LOV2 interface can be engineered to cage other proteins. Engineering and Biology are getting married. If you are a kick ass engineer, consider hacking biology at Ginkgo BioWorks, they are hiring.Wu, Y., Frey, D., Lungu, O., Jaehrig, A., Schlichting, I., Kuhlman, B., & Hahn, K. (2009). A genetically encoded photoactivatable Rac controls the motility of living cells Nature, 461 (7260), 104-108 DOI: 10.1038/nature08241
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Wu, Y., Frey, D., Lungu, O., Jaehrig, A., Schlichting, I., Kuhlman, B., & Hahn, K. (2009) A genetically encoded photoactivatable Rac controls the motility of living cells. Nature, 461(7260), 104-108. DOI: 10.1038/nature08241
- August 28, 2009
- 08:08 AM
- 434 views
[150th post!] GPCR receptor hidden life
by 96well in Reportergene
hidden life of GPCR receptors unveiled with reporter approaches Two recent studies exploited reporter genes to unveil hidden secrets of GPCR signaling which is apparently harder to die than expected. From the cell surface, G-Protein Coupled Receptors are activated by the intended ligand. According to the current feed-back dogma, excessive stimulation results in de-activation (de-sensitization) of the receptor and subsequent internalization.With a genetically-encoded FRET sensor, Païkan Marcaggi and colleagues noted on PNAS that prolonged exposure to a ligand (glutamate) actually increases the sensitivity of the receptor (mGluR) to its ligand, in marked contrast to the desensitization typically observed in such receptors. The group is prototyping a model for receptor activity in which mGluR1 signaling behavior relates primarily to overall duration of glutamate release rather than fluctuations in local neurotransmitter concentration.Following down the feed-back dogma, once internalized, GPCRs are supposed to stop signaling. Davide Calebiro and colleagues recently shared on PLOS Biology the results obtained with a transgenic mouse expressing a fluorescent sensor for GPCRs signaling. By analysing second messengers dynamics, they showed that a GPCR continues to stimulate second messenger production in a sustained manner after internalization.By citing H.P. Rang, Receptor theory is becoming increasingly inadequate as an overall framework for interpreting and analysing drug effects. Studying reporter genes in the context of reporter mice might provide a revolutionary revision in neuroscience, endocrinology and pharmacology.Marcaggi, P., Mutoh, H., Dimitrov, D., Beato, M., & Knopfel, T. (2009). Optical measurement of mGluR1 conformational changes reveals fast activation, slow deactivation, and sensitization Proceedings of the National Academy of Sciences, 106 (27), 11388-11393 DOI: 10.1073/pnas.0901290106Calebiro, D., Nikolaev, V., Gagliani, M., de Filippis, T., Dees, C., Tacchetti, C., Persani, L., & Lohse, M. (2009). Persistent cAMP-Signals Triggered by Internalized G-Protein–Coupled Receptors PLoS Biology, 7 (8) DOI: 10.1371/journal.pbio.1000172
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Marcaggi, P., Mutoh, H., Dimitrov, D., Beato, M., & Knopfel, T. (2009) Optical measurement of mGluR1 conformational changes reveals fast activation, slow deactivation, and sensitization. Proceedings of the National Academy of Sciences, 106(27), 11388-11393. DOI: 10.1073/pnas.0901290106
Calebiro, D., Nikolaev, V., Gagliani, M., de Filippis, T., Dees, C., Tacchetti, C., Persani, L., & Lohse, M. (2009) Persistent cAMP-Signals Triggered by Internalized G-Protein–Coupled Receptors. PLoS Biology, 7(8). DOI: 10.1371/journal.pbio.1000172
- June 14, 2009
- 09:15 AM
- 485 views
Sensitive imaging of T-cells with Gaussia reporter
by 96well in Reportergene
Gaussia luciferase (GLuc) holds the promise to became a great reporter. In the native form, GLuc is secreted. This feature gives additional advantages, but markedly attenuates its application for in vivo imaging. At MSKCC.org, Elmer Santos and colleagues recently described on Nature Medicine a membrane anchored external GLuc (termed extGluc) genetically engineered through the addition of a CD8 transmembrane domain to the carboxy terminus of the enzyme. The strategy to put the reporter outside the cell should be advantageous: the substrate luciferin do not need to enter the cell and variability previously inferred to drug-resistant genes should be avoided. In effect, the new reporter was enough sensitive to monitor in vivo T cells by means of classical bioluminescence imaging on a IVIS workstation.Santos, E., Yeh, R., Lee, J., Nikhamin, Y., Punzalan, B., Punzalan, B., Perle, K., Larson, S., Sadelain, M., & Brentjens, R. (2009). Sensitive in vivo imaging of T cells using a membrane-bound Gaussia princeps luciferase Nature Medicine, 15 (3), 338-344 DOI: 10.1038/nm.1930... Read more »
Santos, E., Yeh, R., Lee, J., Nikhamin, Y., Punzalan, B., Punzalan, B., Perle, K., Larson, S., Sadelain, M., & Brentjens, R. (2009) Sensitive in vivo imaging of T cells using a membrane-bound Gaussia princeps luciferase. Nature Medicine, 15(3), 338-344. DOI: 10.1038/nm.1930
- May 25, 2009
- 04:53 PM
- 573 views
Freedom of robots
by 96well in Reportergene
A Science letter by Robert Stevenson focused my attention on the eventual patentability of new "automated" discoveries. This is of course a letter in response to the "automation of science" previously reviewed. Apparently, it should be legally difficult to patent any invention made by a robot: the American patent law strictly refer to the inventor as "a person", while the European law seems more broad. Thus, supposing a brilliant robot scientist is ever build, no man might protect/deserve those inventions for its proprietary benefit. Legacy is not considered: A invents robot/algorythm B, B invents drug C, but C is not patentable. Honestly, once developed such a robot, it should take no longer to develop a second robot mimicking human creativity (i.e., writing good and bad dates and results on a paper lab-book). Are we so close to Singularity? Interestingly, in a previous Science paper, Debra Meloso from the italian Bocconi University has modeled the patent system and proposed a better way to promote intellectual discovery (maybe including generation of robot-scientists) that should be based on a sort of 2.0 trading of discoveries. Might a machine sell a product? Ask to lawyer Crawford. Stevenson, R., Murphy, J., & Clare, T. (2009). Robot Inventors: Patently Impossible? Science, 324 (5930), 1014-1014 DOI: 10.1126/science.324_1014aMeloso, D., Copic, J., & Bossaerts, P. (2009). Promoting Intellectual Discovery: Patents Versus Markets Science, 323 (5919), 1335-1339 DOI: 10.1126/science.1158624... Read more »
Stevenson, R., Murphy, J., & Clare, T. (2009) Robot Inventors: Patently Impossible?. Science, 324(5930), 1014-1014. DOI: 10.1126/science.324_1014a
Meloso, D., Copic, J., & Bossaerts, P. (2009) Promoting Intellectual Discovery: Patents Versus Markets. Science, 323(5919), 1335-1339. DOI: 10.1126/science.1158624
- May 11, 2009
- 11:04 AM
- 738 views
introducing infrared fluorescence protein IFP
by 96well in Reportergene
In vivo optical imaging of deep tissues in animals is most feasible between 650 and 900 nm because such wavelengths minimize the absorbance by hemoglobin, water, and lipids, as well as light-scattering. Roger Tsien, last year's Nobel Prize in chemistry for his research on fluorescent proteins, introduced in a Science report, a modified version of the Deinococcus radiodurans phytochrome turned to be a infrared fluorescent protein (IFP). Carrying IFP into the mouse liver through an adenovirus-vector, the infrared fluorescence performed better than mKate a red fluorescent protein as imaged by a Maestro spectral imager.This makes me gloomy, since I spoke about mKate in the Reportergene's first post. It was September 2007, does it takes only two years for a reporter gene to be outperformed? It is dramatic: it takes roughly two years to make a transgenic reporter mice, and another two-three years to get data with him! Once you start, you know that you will be outdated at half of your journey.Shu, X., Royant, A., Lin, M., Aguilera, T., Lev-Ram, V., Steinbach, P., & Tsien, R. (2009). Mammalian Expression of Infrared Fluorescent Proteins Engineered from a Bacterial Phytochrome Science, 324 (5928), 804-807 DOI: 10.1126/science.1168683... Read more »
Shu, X., Royant, A., Lin, M., Aguilera, T., Lev-Ram, V., Steinbach, P., & Tsien, R. (2009) Mammalian Expression of Infrared Fluorescent Proteins Engineered from a Bacterial Phytochrome. Science, 324(5928), 804-807. DOI: 10.1126/science.1168683
- May 4, 2009
- 05:23 PM
- 533 views
DNA actively directs transcription itself
by 96well in Reportergene
Traditionally, responsive promoter sequences on DNA have been considered only passive docking sites for a pletora of DNA-binding proteins supposed to play the active hard role of gene expression. Several proteins have been pulled-down according to their ability to bind DNA sequences (i.e., far western blotting) and lot of plasmids were generated carrying any responsive DNA element upstream of a reporter gene to mainly study the activity of such proteins (i.e., transcription factors) and eventually discover new drugs. In other words, reporter assay data have been mainly queried to address gene expression from the protein stand-point (trans-action).It is worth to note, that protein activity is longly known to be allosterically regulated by the binding of ligands or cofactors outside the protein’s active site. Now, Sebastian Meijsing and colleagues from the Yamamoto Lab, are shifting the balance toward cis-acting factors (the DNA itself). They propose in a Science report that DNA is a sequence-specific allosteric ligand for the nuclear receptor GR (glucocorticoid receptor). GR may be considered a ligand-activated transcription factor (i.e., it is activated by cortisol or dexametasone). The Yamamoto group exploited a classic luciferase assay to test the activity resulting from the interaction between GR (protein) on its GREs (DNA) during dexametasone stimulation.Molecular gymnast by:Elio AbbondanzieriInterestingly, odd differences were found in their elegant 2x2 experimental scheme in which either single-poing mutations on the receptor or on the responsive elements were combinatorially tested to finally postulate that DNA topology actively directs transcription similarly to an allosteric regulator. Gene expression , crystallographic, gel-shift and ChIP assays corroborate this intuition. Pay attention to your plasmids: DNA is more than a passive docking site.Meijsing, S., Pufall, M., So, A., Bates, D., Chen, L., & Yamamoto, K. (2009). DNA Binding Site Sequence Directs Glucocorticoid Receptor Structure and Activity Science, 324 (5925), 407-410 DOI: 10.1126/science.1164265... Read more »
Meijsing, S., Pufall, M., So, A., Bates, D., Chen, L., & Yamamoto, K. (2009) DNA Binding Site Sequence Directs Glucocorticoid Receptor Structure and Activity. Science, 324(5925), 407-410. DOI: 10.1126/science.1164265
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