A method for detection of endogenous unmodified RNA in live bacterial cells

RNAs are dynamic molecules that orchestrate a breadth of processes for prokaryotes and eukaryotes in both the nucleus and cytoplasm. These processes include regulation of transcription, translation, and post-translational modifications. It has been demonstrated that the spatio-temporal localization of different RNAs is an important factor in development and the correct localization of some RNAs is crucial for proper development, structural organization and function of the cell. In vivo detection of endogenous RNAs is a challenging task because of low RNA concentrations in live cells, its transient character, limited accessibility for molecular probes, and sensitivity to modification. Methods that study RNA localization utilize either hybridization techniques with pre-labeled probes in fixed cells or require modifications to target RNAs in living cells potentially altering their in vivo behavior. The goal of this project is to design and explore a new non-invasive technique that is capable of detecting unmodified endogenous RNA in living cells. This method utilizes a combination of protein complementation and split aptamer probe technology to fluorescently detect unmodified endogenous RNA in living cells. Experiments show that this approach is capable of detecting a full-length β-globin mRNA in a sequence-dependent manner in live E. coli cells. Most importantly, fluorescent detection of the endogenous inducible phosphate stress response mRNA pstC IS successfully demonstrated. This fluorescent signal Is dependent on the concentration of pstC mRNA. Furthermore, the fluorescent signal revealS a punctate localization of pstC mRNA, which dOES not overlap with nucleoid DNA. This work holds the potential for the next generation of molecular tools for basic RNA research, clinical diagnostics and genetic therapeutics

The Transferrin Receptor Modulates Hfe-Dependent Regulation of Hepcidin Expression

Hemochromatosis is caused by mutations in HFE, a protein that competes with transferrin (TF) for binding to transferrin receptor 1 (TFR1). We developed mutant mouse strains to gain insight into the role of the Hfe/Tfr1 complex in regulating iron homeostasis. We introduced mutations into a ubiquitously expressed Tfr1 transgene or the endogenous Tfr1 locus to promote or prevent the Hfe/Tfr1 interaction. Under conditions favoring a constitutive Hfe/Tfr1 interaction, mice developed iron overload attributable to inappropriately low expression of the hormone hepcidin. In contrast, mice carrying a mutation that interferes with the Hfe/Tfr1 interaction developed iron deficiency associated with inappropriately high hepcidin expression. High-level expression of a liver-specific Hfe transgene in Hfe−/− mice was also associated with increased hepcidin production and iron deficiency. Together, these models suggest that Hfe induces hepcidin expression when it is not in complex with Tfr1

Why Are Outcomes Different for Registry Patients Enrolled Prospectively and Retrospectively? Insights from the Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF).

Background: Retrospective and prospective observational studies are designed to reflect real-world evidence on clinical practice, but can yield conflicting results. The GARFIELD-AF Registry includes both methods of enrolment and allows analysis of differences in patient characteristics and outcomes that may result. Methods and Results: Patients with atrial fibrillation (AF) and ≥1 risk factor for stroke at diagnosis of AF were recruited either retrospectively (n = 5069) or prospectively (n = 5501) from 19 countries and then followed prospectively. The retrospectively enrolled cohort comprised patients with established AF (for a least 6, and up to 24 months before enrolment), who were identified retrospectively (and baseline and partial follow-up data were collected from the emedical records) and then followed prospectively between 0-18 months (such that the total time of follow-up was 24 months; data collection Dec-2009 and Oct-2010). In the prospectively enrolled cohort, patients with newly diagnosed AF (≤6 weeks after diagnosis) were recruited between Mar-2010 and Oct-2011 and were followed for 24 months after enrolment. Differences between the cohorts were observed in clinical characteristics, including type of AF, stroke prevention strategies, and event rates. More patients in the retrospectively identified cohort received vitamin K antagonists (62.1% vs. 53.2%) and fewer received non-vitamin K oral anticoagulants (1.8% vs . 4.2%). All-cause mortality rates per 100 person-years during the prospective follow-up (starting the first study visit up to 1 year) were significantly lower in the retrospective than prospectively identified cohort (3.04 [95% CI 2.51 to 3.67] vs . 4.05 [95% CI 3.53 to 4.63]; p = 0.016). Conclusions: Interpretations of data from registries that aim to evaluate the characteristics and outcomes of patients with AF must take account of differences in registry design and the impact of recall bias and survivorship bias that is incurred with retrospective enrolment. Clinical Trial Registration: - URL: http://www.clinicaltrials.gov . Unique identifier for GARFIELD-AF (NCT01090362)