Highly Parallel Translation of DNA Sequences into Small Molecules

A large body of in vitro evolution work establishes the utility of biopolymer libraries comprising 1010 to 1015 distinct molecules for the discovery of nanomolar-affinity ligands to proteins.[1], [2], [3], [4], [5] Small-molecule libraries of comparable complexity will likely provide nanomolar-affinity small-molecule ligands.[6], [7] Unlike biopolymers, small molecules can offer the advantages of cell permeability, low immunogenicity, metabolic stability, rapid diffusion and inexpensive mass production. It is thought that such desirable in vivo behavior is correlated with the physical properties of small molecules, specifically a limited number of hydrogen bond donors and acceptors, a defined range of hydrophobicity, and most importantly, molecular weights less than 500 Daltons.[8] Creating a collection of 1010 to 1015 small molecules that meet these criteria requires the use of hundreds to thousands of diversity elements per step in a combinatorial synthesis of three to five steps. With this goal in mind, we have reported a set of mesofluidic devices that enable DNA-programmed combinatorial chemistry in a highly parallel 384-well plate format. Here, we demonstrate that these devices can translate DNA genes encoding 384 diversity elements per coding position into corresponding small-molecule gene products. This robust and efficient procedure yields small molecule-DNA conjugates suitable for in vitro evolution experiments

Anti-oestrogens but not oestrogen deprivation promote cellular invasion in intercellular adhesion-deficient breast cancer cells

Introduction Anti-oestrogens have been the mainstay of therapy in patients with oestrogen-receptor (ER) positive breast cancer and have provided significant improvements in survival. However, their benefits are limited by tumour recurrence in a significant proportion of initially drug-responsive breast cancer patients because of acquired anti-oestrogen resistance. Relapse on such therapies clinically presents as local and/or regional recurrences, frequently with distant metastases, and the prognosis for these patients is poor. The selective ER modulator, tamoxifen, classically exerts gene inhibitory effects during the drug-responsive phase in ER-positive breast cancer cells. Paradoxically, this drug is also able to induce the expression of genes, which in the appropriate cell context may contribute to an adverse cell phenotype. Here we have investigated the effects of tamoxifen and fulvestrant treatment on invasive signalling and compared this with the direct effects of oestrogen withdrawal to mimic the action of aromatase inhibitors. Methods The effect of oestrogen and 4-hydroxy-tamoxifen on the invasive capacity of endocrine-sensitive MCF-7 cells, in the presence or absence of functional E-cadherin, was determined by Matrigel invasion assays. Studies also monitored the impact of oestrogen withdrawal or treatment with fulvestrant on cell invasion. Western blotting using phospho-specific antibodies was performed to ascertain changes in invasive signalling in response to the two anti-oestrogens versus both oestradiol treatment and withdrawal. Results To the best of our knowledge, we report for the first time that tamoxifen can promote an invasive phenotype in ER-positive breast cancer cells under conditions of poor cell-cell contact and suggest a role for Src kinase and associated pro-invasive genes in this process. Our studies revealed that although this adverse effect is also apparent for further classes of anti-oestrogens, exemplified by the steroidal agent fulvestrant, it is absent during oestrogen withdrawal. Conclusions These data highlight a previously unreported effect of tamoxifen (and potentially further anti-oestrogens), that such agents appear able to induce breast cancer cell invasion in a specific context (absence of good cell-cell contacts), where these findings may have major clinical implications for those patients with tumours that have inherently poor intercellular adhesion. In such patients oestrogen deprivation with aromatase inhibitors may be more appropriate

An isoperimetric inequality in the plane with a log-convex density

Given a positive lower semi-continuous density $f$ on $\mathbb{R}^2$ the weighted volume $V_f:=f\mathscr{L}^2$ is defined on the $\mathscr{L}^2$-measurable sets in $\mathbb{R}^2$. The $f$-weighted perimeter of a set of finite perimeter $E$ in $\mathbb{R}^2$ is written $P_f(E)$. We study minimisers for the weighted isoperimetric problem $$I_f(v):=\inf\Big\{ P_f(E):E\text{ is a set of finite perimeter in }\mathbb{R}^2\text{ and }V_f(E)=v\Big\}$$ for $v>0$. Suppose $f$ takes the form $f:\mathbb{R}^2\rightarrow(0,+\infty);x\mapsto e^{h(|x|)}$ where $h:[0,+\infty)\rightarrow\mathbb{R}$ is a non-decreasing convex function. Let $v>0$ and $B$ a centred ball in $\mathbb{R}^2$ with $V_f(B)=v$. We show that $B$ is a minimiser for the above variational problem and obtain a uniqueness result

Structural Insights into the Evolution of a Non-Biological Protein: Importance of Surface Residues in Protein Fold Optimization

Phylogenetic profiling of amino acid substitution patterns in proteins has led many to conclude that most structural information is carried by interior core residues that are solvent inaccessible. This conclusion is based on the observation that buried residues generally tolerate only conserved sequence changes, while surface residues allow more diverse chemical substitutions. This notion is now changing as it has become apparent that both core and surface residues play important roles in protein folding and stability. Unfortunately, the ability to identify specific mutations that will lead to enhanced stability remains a challenging problem. Here we discuss two mutations that emerged from an in vitro selection experiment designed to improve the folding stability of a non-biological ATP binding protein. These mutations alter two solvent accessible residues, and dramatically enhance the expression, solubility, thermal stability, and ligand binding affinity of the protein. The significance of both mutations was investigated individually and together, and the X-ray crystal structures of the parent sequence and double mutant protein were solved to a resolution limit of 2.8 and 1.65 Å, respectively. Comparative structural analysis of the evolved protein to proteins found in nature reveals that our non-biological protein evolved certain structural features shared by many thermophilic proteins. This experimental result suggests that protein fold optimization by in vitro selection offers a viable approach to generating stable variants of many naturally occurring proteins whose structures and functions are otherwise difficult to study

Full design automation of multi-state RNA devices to program gene expression using energy-based optimization

[EN] Small RNAs (sRNAs) can operate as regulatory agents to control protein expression by interaction with the 59 untranslated region of the mRNA. We have developed a physicochemical framework, relying on base pair interaction energies, to design multi-state sRNA devices by solving an optimization problem with an objective function accounting for the stability of the transition and final intermolecular states. Contrary to the analysis of the reaction kinetics of an ensemble of sRNAs, we solve the inverse problem of finding sequences satisfying targeted reactions. We show here that our objective function correlates well with measured riboregulatory activity of a set of mutants. This has enabled the application of the methodology for an extended design of RNA devices with specified behavior, assuming different molecular interaction models based on Watson-Crick interaction. We designed several YES, NOT, AND, and OR logic gates, including the design of combinatorial riboregulators. In sum, our de novo approach provides a new paradigm in synthetic biology to design molecular interaction mechanisms facilitating future high-throughput functional sRNA design.Work supported by the grants FP7-ICT-043338 (BACTOCOM) to AJ, and BIO2011-26741 (Ministerio de Economia y Competitividad, Spain) to JAD. GR is supported by an EMBO long-term fellowship co-funded by Marie Curie actions (ALTF-1177-2011), and TEL by a PhD fellowship from the AXA Research Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Rodrigo Tarrega, G.; Landrain, TE.; Majer, E.; Daros Arnau, JA.; Jaramillo, A. (2013). Full design automation of multi-state RNA devices to program gene expression using energy-based optimization. PLoS Computational Biology. 9(8):1003172-1003172. https://doi.org/10.1371/journal.pcbi.1003172S1003172100317298Isaacs, F. J., Dwyer, D. J., & Collins, J. J. (2006). RNA synthetic biology. Nature Biotechnology, 24(5), 545-554. doi:10.1038/nbt1208Isaacs, F. J., Dwyer, D. J., Ding, C., Pervouchine, D. D., Cantor, C. R., & Collins, J. J. (2004). Engineered riboregulators enable post-transcriptional control of gene expression. Nature Biotechnology, 22(7), 841-847. doi:10.1038/nbt986Lucks, J. B., Qi, L., Mutalik, V. K., Wang, D., & Arkin, A. P. (2011). Versatile RNA-sensing transcriptional regulators for engineering genetic networks. Proceedings of the National Academy of Sciences, 108(21), 8617-8622. doi:10.1073/pnas.1015741108Mutalik, V. K., Qi, L., Guimaraes, J. C., Lucks, J. B., & Arkin, A. P. (2012). Rationally designed families of orthogonal RNA regulators of translation. Nature Chemical Biology, 8(5), 447-454. doi:10.1038/nchembio.919Bayer, T. S., & Smolke, C. D. (2005). Programmable ligand-controlled riboregulators of eukaryotic gene expression. Nature Biotechnology, 23(3), 337-343. doi:10.1038/nbt1069Nakashima, N., & Tamura, T. (2009). Conditional gene silencing of multiple genes with antisense RNAs and generation of a mutator strain of Escherichia coli. Nucleic Acids Research, 37(15), e103-e103. doi:10.1093/nar/gkp498Callura, J. M., Cantor, C. R., & Collins, J. J. (2012). Genetic switchboard for synthetic biology applications. Proceedings of the National Academy of Sciences, 109(15), 5850-5855. doi:10.1073/pnas.1203808109Beisel, C. L., Bayer, T. S., Hoff, K. G., & Smolke, C. D. (2008). Model‐guided design of ligand‐regulated RNAi for programmable control of gene expression. Molecular Systems Biology, 4(1), 224. doi:10.1038/msb.2008.62Qi, L., Lucks, J. B., Liu, C. C., Mutalik, V. K., & Arkin, A. P. (2012). Engineering naturally occurring trans -acting non-coding RNAs to sense molecular signals. Nucleic Acids Research, 40(12), 5775-5786. doi:10.1093/nar/gks168Carothers, J. M., Goler, J. A., Juminaga, D., & Keasling, J. D. (2011). Model-Driven Engineering of RNA Devices to Quantitatively Program Gene Expression. Science, 334(6063), 1716-1719. doi:10.1126/science.1212209Rodrigo, G., Landrain, T. E., & Jaramillo, A. (2012). De novo automated design of small RNA circuits for engineering synthetic riboregulation in living cells. Proceedings of the National Academy of Sciences, 109(38), 15271-15276. doi:10.1073/pnas.1203831109Brantl, S. (2002). Antisense-RNA regulation and RNA interference. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1575(1-3), 15-25. doi:10.1016/s0167-4781(02)00280-4Majdalani, N., Vanderpool, C. K., & Gottesman, S. (2005). Bacterial Small RNA Regulators. Critical Reviews in Biochemistry and Molecular Biology, 40(2), 93-113. doi:10.1080/10409230590918702Selinger, D. W., Cheung, K. J., Mei, R., Johansson, E. M., Richmond, C. S., Blattner, F. R., … Church, G. M. (2000). RNA expression analysis using a 30 base pair resolution Escherichia coli genome array. Nature Biotechnology, 18(12), 1262-1268. doi:10.1038/82367Yelin, R., Dahary, D., Sorek, R., Levanon, E. Y., Goldstein, O., Shoshan, A., … Rotman, G. (2003). Widespread occurrence of antisense transcription in the human genome. Nature Biotechnology, 21(4), 379-386. doi:10.1038/nbt808Wang, X.-J., Gaasterland, T., & Chua, N.-H. (2005). Genome Biology, 6(4), R30. doi:10.1186/gb-2005-6-4-r30Stojanovic, M. N., & Stefanovic, D. (2003). A deoxyribozyme-based molecular automaton. Nature Biotechnology, 21(9), 1069-1074. doi:10.1038/nbt862Seelig, G., Soloveichik, D., Zhang, D. Y., & Winfree, E. (2006). Enzyme-Free Nucleic Acid Logic Circuits. Science, 314(5805), 1585-1588. doi:10.1126/science.1132493Yin, P., Choi, H. M. T., Calvert, C. R., & Pierce, N. A. (2008). Programming biomolecular self-assembly pathways. Nature, 451(7176), 318-322. doi:10.1038/nature06451Ran, T., Kaplan, S., & Shapiro, E. (2009). Molecular implementation of simple logic programs. Nature Nanotechnology, 4(10), 642-648. doi:10.1038/nnano.2009.203Penchovsky, R., & Breaker, R. R. (2005). Computational design and experimental validation of oligonucleotide-sensing allosteric ribozymes. Nature Biotechnology, 23(11), 1424-1433. doi:10.1038/nbt1155Salis, H. M., Mirsky, E. A., & Voigt, C. A. (2009). Automated design of synthetic ribosome binding sites to control protein expression. Nature Biotechnology, 27(10), 946-950. doi:10.1038/nbt.1568Laidler, K. J., & King, M. C. (1983). Development of transition-state theory. The Journal of Physical Chemistry, 87(15), 2657-2664. doi:10.1021/j100238a002Sosnick, T. R., & Pan, T. (2003). RNA folding: models and perspectives. Current Opinion in Structural Biology, 13(3), 309-316. doi:10.1016/s0959-440x(03)00066-6Yurke, B. (2003). Genetic Programming and Evolvable Machines, 4(2), 111-122. doi:10.1023/a:1023928811651Bandyra, K. J., Said, N., Pfeiffer, V., Górna, M. W., Vogel, J., & Luisi, B. F. (2012). The Seed Region of a Small RNA Drives the Controlled Destruction of the Target mRNA by the Endoribonuclease RNase E. Molecular Cell, 47(6), 943-953. doi:10.1016/j.molcel.2012.07.015Dawid, A., Cayrol, B., & Isambert, H. (2009). RNA synthetic biology inspired from bacteria: construction of transcription attenuators under antisense regulation. Physical Biology, 6(2), 025007. doi:10.1088/1478-3975/6/2/025007Lioliou, E., Romilly, C., Romby, P., & Fechter, P. (2010). RNA-mediated regulation in bacteria: from natural to artificial systems. New Biotechnology, 27(3), 222-235. doi:10.1016/j.nbt.2010.03.002Dirks, R. M., Bois, J. S., Schaeffer, J. M., Winfree, E., & Pierce, N. A. (2007). Thermodynamic Analysis of Interacting Nucleic Acid Strands. SIAM Review, 49(1), 65-88. doi:10.1137/060651100Das, R., Karanicolas, J., & Baker, D. (2010). Atomic accuracy in predicting and designing noncanonical RNA structure. Nature Methods, 7(4), 291-294. doi:10.1038/nmeth.1433Vogel, J., & Luisi, B. F. (2011). Hfq and its constellation of RNA. Nature Reviews Microbiology, 9(8), 578-589. doi:10.1038/nrmicro2615Friedland, A. E., Lu, T. K., Wang, X., Shi, D., Church, G., & Collins, J. J. (2009). Synthetic Gene Networks That Count. Science, 324(5931), 1199-1202. doi:10.1126/science.1172005Rodrigo, G., Carrera, J., Landrain, T. E., & Jaramillo, A. (2012). Perspectives on the automatic design of regulatory systems for synthetic biology. FEBS Letters, 586(15), 2037-2042. doi:10.1016/j.febslet.2012.02.031Chin, J. W. (2006). Modular approaches to expanding the functions of living matter. Nature Chemical Biology, 2(6), 304-311. doi:10.1038/nchembio789McCaskill, J. S. (1990). The equilibrium partition function and base pair binding probabilities for RNA secondary structure. Biopolymers, 29(6-7), 1105-1119. doi:10.1002/bip.360290621Chitsaz, H., Salari, R., Sahinalp, S. C., & Backofen, R. (2009). A partition function algorithm for interacting nucleic acid strands. Bioinformatics, 25(12), i365-i373. doi:10.1093/bioinformatics/btp212Hofacker, I. L., Fontana, W., Stadler, P. F., Bonhoeffer, L. S., Tacker, M., & Schuster, P. (1994). Fast folding and comparison of RNA secondary structures. Monatshefte f�r Chemie Chemical Monthly, 125(2), 167-188. doi:10.1007/bf00818163Andronescu, M., Zhang, Z. C., & Condon, A. (2005). Secondary Structure Prediction of Interacting RNA Molecules. Journal of Molecular Biology, 345(5), 987-1001. doi:10.1016/j.jmb.2004.10.082Mathews, D. H., Sabina, J., Zuker, M., & Turner, D. H. (1999). Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. Journal of Molecular Biology, 288(5), 911-940. doi:10.1006/jmbi.1999.2700Kirkpatrick, S., Gelatt, C. D., & Vecchi, M. P. (1983). Optimization by Simulated Annealing. Science, 220(4598), 671-680. doi:10.1126/science.220.4598.67

Secret worlds, democratization and election observation in Malawi

ASC – Publicaties niet-programma gebonde

Low-temperature zircon growth related to hydrothermal alteration of siderite concretions in Mississippian shales, Scotland

Zircon occurs in voids and cracks in phosphatic coprolites enclosed in siderite concretions in Mississippian shales near Edinburgh, Scotland. The zircon formed during hydrothermal alteration of early-diagenetic concretions and occurs as spherical aggregates of prismatic crystals, sometimes radiating. Vitrinite reflectance measurements indicate temperatures of ~270°C for the zircon-bearing concretions and the host shales. Molecular parameter values based on dibenzothiophene and phenanthrene distribution and occurrence of di- and tetra-hydro-products of polycyclic aromatic compounds suggest that the rocks experienced relatively high-temperature aqueous conditions related to hydrothermal fluids, perhaps associated with neighboring mafic intrusions. The zircon was dissolved from the concretions, transported in fluids, and reprecipitated in voids. This is the first record of the precipitation of authigenic zircon in sedimentary rock as a new phase, not as outgrowths