Synthetic gene networks can be constructed to emulate digital circuits and devices, giving one the ability to program and design cells with some of the principles of modern computing, such as counting. A cellular counter would enable complex synthetic programming and a variety of biotechnology applications. Here we report two complementary synthetic genetic counters in E. coli that can count up to three induction events, the first comprised of a riboregulated transcriptional cascade and the second of a recombinase-based cascade of memory units. These modular devices permit counting of varied user-defined inputs over a range of frequencies and can be expanded to count higher numbers.A counter is a key component in digital circuits and computing that retains memory of events or objects, representing each number of such as a distinct state. Counters would also be useful in cells, which often must have accurate accounting of tightly controlled processes or biomolecules to effectively maintain metabolism and growth. Counting mechanisms have been reportedly found in telomere length regulation (1,2) and cell aggregation (3). These system behaviors appear to be the result of a thresholding effect in which some critical molecule number or density must be reached for the observed phenotypic change.In this study, we first develop a counter, termed the Riboregulated Transcriptional Cascade (RTC) Counter, which is based on a transcriptional cascade with additional translational regulation. Figs. 1A and 1C illustrate two such cascades that can count up to 2 and 3, respectively (hence, the designations RTC 2-Counter and RTC 3-Counter). For the RTC 2-Counter, the constitutive promoter P Ltet0-1 drives transcription of T7 RNA polymerase (RNAP), whose protein binds the T7 promoter and transcribes the downstream gene, in this case Green Fluorescent Protein (GFP). Both genes are additionally regulated by riboregulators (4), whose cis and trans elements silence and activate post-transcriptional gene expression, respectively. The cis-repressor sequence (cr) is placed between the transcription start site and the ribosome binding site (RBS), and its complementarity with the RBS causes a stem-loop structure to form upon transcription. This secondary structure prevents binding of the 30S ribosomal subunit to the RBS, inhibiting translation. A short, trans-activating, noncoding RNA (taRNA) driven by the arabinose promoter P BAD binds to the cis-repressor in trans, relieving * This manuscript has been accepted for publication in Science. This version has not undergone final editing. Please refer to the complete version of record at http://www.sciencemag.org/. The manuscript may not be reproduced or used in any manner that does not fall within the fair use provisions of the Copyright Act without the prior, written permission of AAAS. * These authors contributed equally to this work. 1A). With cis-repressed T7 RNAP mRNAs in the cell, the first pulse of arabinose drives a short burst of taRNA production and consequently expression of...
Mechanical and thermal stimuli acting on the skin are detected by morphologically and physiologically distinct sensory neurons of the dorsal root ganglia (DRG). Achieving a holistic view of how this diverse neuronal population relays sensory information from the skin to the central nervous system (CNS) has been challenging with existing tools. Here, we used transcriptomic datasets of the mouse DRG to guide development and curation of a genetic toolkit to interrogate transcriptionally defined DRG neuron subtypes. Morphological analysis revealed unique cutaneous axon arborization areas and branching patterns of each subtype. Physiological analysis showed that subtypes exhibit distinct thresholds and ranges of responses to mechanical and/or thermal stimuli. The somatosensory neuron toolbox thus enables comprehensive phenotyping of most principal sensory neuron subtypes. Moreover, our findings support a population coding scheme in which the activation thresholds of morphologically and physiologically distinct cutaneous DRG neuron subtypes tile multiple dimensions of stimulus space.
Sickle Cell Disease (SCD) is characterized by hemolytic anemia, which can trigger oxidative stress, inflammation, and tissue injury that contributes to disease complications. Bone marrow mesenchymal stromal cells (MSCs) tightly regulate hematopoietic stem cell (HSC) homeostasis in health and disease but their functionality in SCD remains unclear. We identified for the first time murine SCD MSCs to have altered gene signatures, reduced stem cell properties, and increased oxidative stress, due in part to hemolysis. Murine SCD MSCs had lower HSC maintenance ability in vitro and in vivo as manifested by increased HSC mobilization and decreased HSC engraftment following transplant. Activation of TLR4 through p65 in MSCs further contributed to MSC dysfunction. Transfusions led to improved MSC and HSC oxidative state in SCD mice. Improving the regulation between MSCs and HSCs has vital implications for enhancing clinical HSC transplantation and gene therapy outcomes and for identification of new molecular targets for alleviating SCD complications.
Mediastinal mass syndrome (MMS) is a complex case that poses many challenges to the anesthesiologist. The cornerstone of management focuses on the potential hemodynamic changes associated with this syndrome. We describe the anesthetic management of a patient with a previously undiagnosed mediastinal mass presenting for emergency neurosurgical surgery.
In analyzing the thought of the 1930s, historians have usually concentrated on the reactions of various liberal and leftist critics of Roosevelt's New Deal reform programs. A few, however, have stressed that in opposition both to the New Deal's regulated welfare capitalism and to the left's many-faceted demands for a more openly radical program, the period also witnessed some significant theorizing that remained conservative and even reactionary. Considerable attention, for instance, has been given to the Nashville Agrarians who in November 1930 published I'll Take My Stand, their ardent manifesto of Southern cultural independence from the capitalistic, urban-industrial north. Yet it is not so well known that the Jeffersonian social theory they espoused was the basis for a broader, national effort to promote the decentralization of American government and industry and to foster greater economic self-sufficiency through a wider distribution of property. For ten years, 1937–1947, the journal Free America served as an organ for this “decentralist” movement.
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