Predictive RNA profiles for early and very early spontaneous preterm birth.

BACKGROUND: Spontaneous preterm birth remains the main driver of childhood morbidity and mortality. Because of an incomplete understanding of the molecular pathways that result in spontaneous preterm birth, accurate predictive markers and target therapeutics remain elusive. OBJECTIVE: This study sought to determine if a cell-free RNA profile could reveal a molecular signature in maternal blood months before the onset of spontaneous preterm birth. STUDY DESIGN: Maternal samples (n=242) were obtained from a prospective cohort of individuals with a singleton pregnancy across 4 clinical sites at 12-24 weeks (nested case-control; n=46 spontaneous preterm birth <35 weeks and n=194 term controls). Plasma was processed via a next-generation sequencing pipeline for cell-free RNA using the Mirvie RNA platform. Transcripts that were differentially expressed in next-generation sequencing cases and controls were identified. Enriched pathways were identified in the Reactome database using overrepresentation analysis. RESULTS: Twenty five transcripts associated with an increased risk of spontaneous preterm birth were identified. A logistic regression model was developed using these transcripts to predict spontaneous preterm birth with an area under the curve =0.80 (95% confidence interval, 0.72-0.87) (sensitivity=0.76, specificity=0.72). The gene discovery and model were validated through leave-one-out cross-validation. A unique set of 39 genes was identified from cases of very early spontaneous preterm birth (<25 weeks, n=14 cases with time to delivery of 2.5±1.8 weeks); a logistic regression classifier on the basis of these genes yielded an area under the curve=0.76 (95% confidence interval, 0.63-0.87) in leave-one-out cross validation. Pathway analysis for the transcripts associated with spontaneous preterm birth revealed enrichment of genes related to collagen or the extracellular matrix in those who ultimately had a spontaneous preterm birth at <35 weeks. Enrichment for genes in insulin-like growth factor transport and amino acid metabolism pathways were associated with spontaneous preterm birth at <25 weeks. CONCLUSION: Second trimester cell-free RNA profiles in maternal blood provide a noninvasive window to future occurrence of spontaneous preterm birth. The systemic finding of changes in collagen and extracellular matrix pathways may serve to identify individuals at risk for premature cervical remodeling, with growth factor and metabolic pathways implicated more often in very early spontaneous preterm birth. The use of cell-free RNA profiles has the potential to accurately identify those at risk for spontaneous preterm birth by revealing the underlying pathophysiology, creating an opportunity for more targeted therapeutics and effective interventions.

RNA profiles reveal signatures of future health and disease in pregnancy.

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.

SLLISWD sequence in the 10FNIII domain initiates fibronectin fibrillogenesis.

Fibronectin (FN) assembly into extracellular matrix is tightly regulated and essential to embryogenesis and wound healing. FN fibrillogenesis is initiated by cytoskeleton-derived tensional forces transmitted across transmembrane integrins onto RGD binding sequences within the tenth FN type III (10FNIII) domains. These forces unfold 10FNIII to expose cryptic FN assembly sites; however, a specific sequence has not been identified in 10FNIII. Our past steered molecular dynamics simulations modeling 10FNIII unfolding by force at its RGD loop predicted a mechanical intermediate with a solvent-exposed N terminus spanning the A and B ß-strands. Here, we experimentally confirm that the predicted 23-residue cryptic peptide 1 (CP1) initiates FN multimerization, which is mediated by interactions with 10FNIII that expose hydrophobic surfaces that support 8-anilino-1-napthalenesulfonic acid binding. Localization of multimerization activity to the C terminus led to the discovery of a minimal 7-amino acid "multimerization sequence" (SLLISWD), which induces polymerization of FN and the clotting protein fibrinogen in addition to enhancing FN fibrillogenesis in fibroblasts. A point mutation at Trp-6 that reduces exposure of hydrophobic sites for 8-anilino-1-napthalenesulfonic acid binding and ß-structure formation inhibits FN multimerization and prevents physiological cell-based FN assembly in culture. We propose a model for cell-mediated fibrillogenesis whereby cell traction force initiates a cascade of intermolecular exchange starting with the unfolding of 10FNIII to expose the multimerization sequence, which interacts with strand B of another 10FNIII domain via a Trp-mediated ß-strand exchange to stabilize a partially unfolded intermediate that propagates FN self-assembly.

Control of lung vascular permeability and endotoxin-induced pulmonary oedema by changes in extracellular matrix mechanics.

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-α. 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.

Fibronectin unfolding revisited: modeling cell traction-mediated unfolding of the tenth type-III repeat.

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 10(th) 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 beta-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.

Rational design of memory in eukaryotic cells.

The ability to logically engineer novel cellular functions promises a deeper understanding of biological systems. Here we demonstrate the rational design of cellular memory in yeast that employs autoregulatory transcriptional positive feedback. We built a set of transcriptional activators and quantitatively characterized their effects on gene expression in living cells. Modeling in conjunction with the quantitative characterization of the activator-promoter pairs accurately predicts the behavior of the memory network. This study demonstrates the power of taking advantage of components with measured quantitative parameters to specify eukaryotic regulatory networks with desired properties.