Evaluation of acrylamide-based molecularly imprinted polymer thin-sheets for specific protein capture - a myoglobin model

We evaluate a series of thin-sheet hydrogel molecularly imprinted polymers (MIPs), using a family of acrylamide-based monomers, selective for the target protein myoglobin (Mb). The simple production of the thin-sheet MIP offers an alternative biorecognition surface that is robust, stable and uniform, and has the potential to be adapted for biosensor applications. The MIP containing the functional monomer N-hydroxymethylacrylamide (NHMAm), produced optimal specific rebinding of the target protein (Mb) with 84.9 % (± 0.7) rebinding and imprinting and selectivity factors of 1.41 and 1.55, respectively. The least optimal performing MIP contained the functional monomer N,Ndimethylacrylamide (DMAm) with 67.5 % (± 0.7) rebinding and imprinting and selectivity factors of 1.11 and 1.32, respectively. Hydrogen bonding effects, within a protein-MIP complex, were investigated using computational methods and Fourier transform infrared (FTIR) spectroscopy. The quantum mechanical calculations predictions of a red shift of the monomer carbonyl peak is borneout within FTIR spectra, with three of the MIPs, acrylamide, N-(hydroxymethyl) acrylamide, and N-(hydroxyethyl) acrylamide, showing peak downshifts of 4, 11, and 8 cm-1, respectively

Linear DNA for Rapid Prototyping of Synthetic Biological Circuits in an Escherichia coli Based TX-TL Cell-Free System

Accelerating the pace of synthetic biology experiments requires new approaches for rapid prototyping of circuits from individual DNA regulatory elements. However, current testing standards require days to weeks due to cloning and in vivo transformation. In this work, we first characterized methods to protect linear DNA strands from exonuclease degradation in an Escherichia coli based transcription-translation cell-free system (TX-TL), as well as mechanisms of degradation. This enabled the use of linear DNA PCR products in TX-TL. We then compared expression levels and binding dynamics of different promoters on linear DNA and plasmid DNA. We also demonstrated assembly technology to rapidly build circuits entirely in vitro from separate parts. Using this strategy, we prototyped a four component genetic switch in under 8 h entirely in vitro. Rapid in vitro assembly has future applications for prototyping multiple component circuits if combined with predictive computational models

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform. This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology. To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems. The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted. The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters. Accompanying mathematical models are available. The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard.

Rapidly Characterizing the Fast Dynamics of RNA Genetic Circuitry with Cell-Free Transcription Translation (TX-TL) Systems

RNA regulators are emerging as powerful tools to engineer synthetic genetic networks or rewire existing ones. A potential strength of RNA networks is that they may be able to propagate signals on time scales that are set by the fast degradation rates of RNAs. However, a current bottleneck to verifying this potential is the slow design-build-test cycle of evaluating these networks in vivo. Here, we adapt an Escherichia coli-based cell-free transcription-translation (TX-TL) system for rapidly prototyping RNA networks. We used this system to measure the response time of an RNA transcription cascade to be approximately five minutes per step of the cascade. We also show that this response time can be adjusted with temperature and regulator threshold tuning. Finally, we use TX-TL to prototype a new RNA network, an RNA single input module, and show that this network temporally stages the expression of two genes in vivo

Consensus on circulatory shock and hemodynamic monitoring. Task force of the European Society of Intensive Care Medicine.

OBJECTIVE: Circulatory shock is a life-threatening syndrome resulting in multiorgan failure and a high mortality rate. The aim of this consensus is to provide support to the bedside clinician regarding the diagnosis, management and monitoring of shock. METHODS: The European Society of Intensive Care Medicine invited 12 experts to form a Task Force to update a previous consensus (Antonelli et al.: Intensive Care Med 33:575-590, 2007). The same five questions addressed in the earlier consensus were used as the outline for the literature search and review, with the aim of the Task Force to produce statements based on the available literature and evidence. These questions were: (1) What are the epidemiologic and pathophysiologic features of shock in the intensive care unit ? (2) Should we monitor preload and fluid responsiveness in shock ? (3) How and when should we monitor stroke volume or cardiac output in shock ? (4) What markers of the regional and microcirculation can be monitored, and how can cellular function be assessed in shock ? (5) What is the evidence for using hemodynamic monitoring to direct therapy in shock ? Four types of statements were used: definition, recommendation, best practice and statement of fact. RESULTS: Forty-four statements were made. The main new statements include: (1) statements on individualizing blood pressure targets; (2) statements on the assessment and prediction of fluid responsiveness; (3) statements on the use of echocardiography and hemodynamic monitoring. CONCLUSIONS: This consensus provides 44 statements that can be used at the bedside to diagnose, treat and monitor patients with shock

Characterizing and prototyping genetic networks with cell-free transcription–translation reactions

A central goal of synthetic biology is to engineer cellular behavior by engineering synthetic gene networks for a variety of biotechnology and medical applications. The process of engineering gene networks often involves an iterative ‘design–build–test’ cycle, whereby the parts and connections that make up the network are built, characterized and varied until the desired network function is reached. Many advances have been made in the design and build portions of this cycle. However, the slow process of in vivo characterization of network function often limits the timescale of the testing step. Cell-free transcription–translation (TX–TL) systems offer a simple and fast alternative to performing these characterizations in cells. Here we provide an overview of a cell-free TX–TL system that utilizes the native Escherichia coli TX–TL machinery, thereby allowing a large repertoire of parts and networks to be characterized. As a way to demonstrate the utility of cell-free TX–TL, we illustrate the characterization of two genetic networks: an RNA transcriptional cascade and a protein regulated incoherent feed-forward loop. We also provide guidelines for designing TX–TL experiments to characterize new genetic networks. We end with a discussion of current and emerging applications of cell free systems

Topographically Influenced Evolution of Large-scale Changes in Comet 67P/Churyumov-Gerasimenko's Imhotep Region

Large portions of comet 67P/Churyumov–Gerasimenko’s northern hemisphere are blanketed by fallback material consisting of centimeter-sized particles termed the smooth terrains. Observations from the Rosetta mission show that the most drastic transient changes during 67P’s 2015 perihelion passage occurred within a subset of these deposits. However, we still do not understand the processes driving these changes, limiting our overall understanding of how comets evolve over both seasonal and multiorbit timescales. Herein we provide a complete documentation of scarp-driven activity on 67P’s largest smooth terrain deposit, a highly active portion of the Imhotep region that is the southernmost of all smooth terrain basins on 67P. We also present a thermal model that accurately predicts when and where scarps originate during the course of the observed activity. Assuming a uniform surface composition, our model shows that activity is heavily controlled by local topography rather than the presence of ice-enhanced hot spots on the surface. Scarps within the smooth terrain deposits in central Imhotep also exhibit a peculiar behavior, where three scarps originate from the same location but at different times and migrate in opposite directions. This behavior indicates that the landscape retains a memory of previous cycles of erosion and deposition, reflected by the depth of the volatile-rich layer. Future work will need to couple our thermal model with a landscape evolution model in order to explain the complete dynamic evolution of these terrains