Quantifying the spatiotemporal dynamics of IRES versus Cap translation with single-molecule resolution in living cells

ABSTRACTViruses use IRES sequences within their RNA to hijack translation machinery and thereby rapidly replicate in host cells. While this process has been extensively studied in bulk assays, the dynamics of hijacking at the single-molecule level remain unexplored in living cells. To achieve this, we developed a bicistronic biosensor encoding complementary repeat epitopes in two ORFs, one translated in a Cap-dependent manner and the other translated in an IRES-mediated manner. Using a pair of complementary probes that bind the epitopes co-translationally, our biosensor lights up in different colors depending on which ORF is being translated. In combination with single-molecule tracking and computational modeling, we measured the relative kinetics of Cap versus IRES translation and show: (1) Two non-overlapping ORFs can be simultaneously translated within a single mRNA; (2) EMCV IRES-mediated translation sites recruit ribosomes less efficiently than Cap-dependent translation sites but are otherwise nearly indistinguishable, having similar mobilities, sizes, spatial distributions, and ribosomal initiation and elongation rates; (3) Both Cap-dependent and IRES-mediated ribosomes tend to stretch out translation sites; (4) Although the IRES recruits two to three times fewer ribosomes than the Cap in normal conditions, the balance shifts dramatically in favor of the IRES during oxidative and ER stresses that mimic viral infection; and (5) Translation of the IRES is enhanced by translation of the Cap, demonstrating upstream translation can positively impact the downstream translation of a non-overlapping ORF. With the ability to simultaneously quantify two distinct translation mechanisms in physiologically relevant live-cell environments, we anticipate bicistronic biosensors like the one we developed here will become powerful new tools to dissect both canonical and non-canonical translation dynamics with single-molecule precision.Graphical Abstract

Myeloid-derived suppressor cells in secondary sepsis: Is there an association with lethal outcome?

Background/Aim. Role of myeloid-derived suppressor cells (MDSCs) in human host response to sepsis still needs to be clarified. The aim of our study was to determine whether frequency and/or absolute numbers of the MDSCs were associated with outcome in critically ill patients with secondary sepsis and/or septic shock. Methods. Total of 40 critically ill patients with secondary sepsis were enrolled in a prospective study. We detected and enumerated both main subsets of MDSCs: granulocytic (G)-MDSCs and monocytic (M)- MDSCs on the Day 1 (the day of hospital admission) and the Day 5 after the. The primary end-point was hospital mortality. Results. Increased frequencies and absolute numbers of subpopulations corresponding to MDSCs were associated with poor outcome. As far as relative kinetics was concerned, in both survivors and non-survivors, sepsis duration from 1th to 5th day was accompanied by an increase in MDSCs values of both investigated subpopulations. In contrast to findings of stepwise multivariate logistic regression analysis of the variables on the Day 1, on the Day 5 it was determined that the Sequential Organ Failure Assessment (SOFA) score (OR 2.350; p < 0.05) and G-MDSCs frequencies (OR 3.575; p < 0.05) were independent predictors of lethal outcome. Conclusion. These findings suggest harmful role of MDSCs in secondary sepsis.

A one-pot strategy to improve end-capping efficacy in Stille poly-condensations

A reproducible synthetic method affording end-functionalized conjugated polymers via Stille polycondensations based upon end-capping reagent relative kinetics is presented.