The field of synthetic biology aims to design biological systems to perform tasks to better understand analogous natural systems and for direct applications in research and medicine (e.g., see Andrianantoandro et al. 2006;Drubin et al. 2007). Currently our ability to design biological systems is limited by the difficulty of predicting the behavior of even simple genetic circuits because often a given network topology will show qualitatively different behavior depending on the quantitative features of the underlying components. While advances have been made with networks composed of well-studied modules in bacteria (Guido et al. 2006), this challenge is acute in more complex eukaryotic systems where the components rarely have measured characteristics. Therefore, to make rapid progress in designing eukaryotic systems, synthetic biologists require both a pool of quantitatively annotated biological parts and the knowledge that these parts can be logically engineered into more complex networks with predictable function.One such network, which carries intrinsic value and tests a bottom-up design approach, is a network that confers memory. In this study we describe the rational design and construction of a high fidelity, modular memory device in yeast based on transcriptionally controlled autoregulatory positive feedback. This device heritably retains an induced state in individual cells in response to a transient stimulus. The rational design approach used here employs an extensive in vivo quantitative characterization of a set of synthetic transcription factors and the prediction of system behavior via network models incorporating these measured parameters. By successfully constructing this memory device, we established the essential parameters for maintaining an autoregulatory positive feedback loop in a dividing cellular system. Most importantly, we demonstrated predictability of system behavior in eukaryotes when the system is built from well-understood components. Results and Discussion Functional activators based on a modular architectureTo rationally engineer a memory device, we designed a set of fluorescently labeled synthetic transcription factors and their corresponding reporter genes to serve as candidate components. Each activator gene consists of a DNA-binding domain (DBD), two tandem copies of the monomeric red fluorescent protein (RFP) mCherry (Shaner et al. 2004), the viral activation domain VP64 (Beerli et al. 1998), and the SV40 nuclear localization sequence (NLS) (Kalderon et al. 1984;Lanford and Butel 1984), all under control of the GAL1/10 promoter (Fig. 1A). Each reporter gene has multiple copies of the DNAbinding sites corresponding to its given transcription factor upstream of the minimal CYC1 promoter, and its protein coding region encodes two tandem copies of the yellow fluorescent protein variant (YFP) Venus ( Fig. 1A; Nagai et al. 2002). The DBDs used were the LexA DBD (Hurstel et al. 1986(Hurstel et al. , 1988, an engineered variant of the murine zinc-finger Zif268 (ZifH) (Hurt et al. 200...

Increased vascular permeability contributes to many diseases, including acute respiratory distress syndrome, cancer and inflammation. Most past work on vascular barrier function has focused on soluble regulators, such as tumour-necrosis factor-a. Here we show that lung vascular permeability is controlled mechanically by changes in extracellular matrix structure. Our studies reveal that pulmonary vascular leakage can be increased by altering extracellular matrix compliance in vitro and by manipulating lysyl oxidase-mediated collagen crosslinking in vivo. Either decreasing or increasing extracellular matrix stiffness relative to normal levels disrupts junctional integrity and increases vascular leakage. Importantly, endotoxin-induced increases of vascular permeability are accompanied by concomitant increases in extracellular matrix rigidity and lysyl oxidase activity, which can be prevented by inhibiting lysyl oxidase activity. The identification of lysyl oxidase and the extracellular matrix as critical regulators of lung vascular leakage might lead to the development of new therapeutic approaches for the treatment of pulmonary oedema and other diseases caused by abnormal vascular permeability.

Maternal morbidity and mortality continue to rise, and pre-eclampsia is a major driver of this burden1. Yet the ability to assess underlying pathophysiology before clinical presentation to enable identification of pregnancies at risk remains elusive. Here we demonstrate the ability of plasma cell-free RNA (cfRNA) to reveal patterns of normal pregnancy progression and determine the risk of developing pre-eclampsia months before clinical presentation. Our results centre on comprehensive transcriptome data from eight independent prospectively collected cohorts comprising 1,840 racially diverse pregnancies and retrospective analysis of 2,539 banked plasma samples. The pre-eclampsia data include 524 samples (72 cases and 452 non-cases) from two diverse independent cohorts collected 14.5 weeks (s.d., 4.5 weeks) before delivery. We show that cfRNA signatures from a single blood draw can track pregnancy progression at the placental, maternal and fetal levels and can robustly predict pre-eclampsia, with a sensitivity of 75% and a positive predictive value of 32.3% (s.d., 3%), which is superior to the state-of-the-art method2. cfRNA signatures of normal pregnancy progression and pre-eclampsia are independent of clinical factors, such as maternal age, body mass index and race, which cumulatively account for less than 1% of model variance. Further, the cfRNA signature for pre-eclampsia contains gene features linked to biological processes implicated in the underlying pathophysiology of pre-eclampsia.

Fibronectin polymerization is essential for the development and repair of the extracellular matrix. Consequently, deciphering the mechanism of fibronectin fibril formation is of immense interest. Fibronectin fibrillogenesis is driven by cell-traction forces that mechanically unfold particular modules within fibronectin. Previously, mechanical unfolding of fibronectin has been modeled by applying tensile forces at the N- and C-termini of fibronectin domains; however, physiological loading is likely focused on the solvent-exposed RGD loop in the 10th type-III repeat of fibronectin (10FNIII), which mediates binding to cell-surface integrin receptors. In this work we used steered molecular dynamics to study the mechanical unfolding of 10FNIII under tensile force applied at this RGD site. We demonstrate that mechanically unfolding 10FNIII by pulling at the RGD site requires less work than unfolding by pulling at the N- and C- termini. Moreover, pulling at the N- and C-termini leads to 10FNIII unfolding along several pathways while pulling on the RGD site leads to a single exclusive unfolding pathway that includes a partially unfolded intermediate with exposed hydrophobic N-terminal β-strands – residues that may facilitate fibronectin self-association. Additional mechanical unfolding triggers an essential arginine residue, which is required for high affinity binding to integrins, to move to a position far from the integrin binding site. This cell traction-induced conformational change may promote cell detachment after important partially unfolded kinetic intermediates are formed. These data suggest a novel mechanism that explains how cell-mediated forces promote fibronectin fibrillogenesis and how cell surface integrins detach from newly forming fibrils. This process enables cells to bind and unfold additional fibronectin modules – a method that propagates matrix assembly.