Establishing Site-Directed A-to-I RNA Editing in Cell Culture

Tools to manipulate genetic information without interfering at the DNA level are highly desirable in medicine and the life sciences. Recently, our group introduced the first engineered, RNA-guided deaminase. The approach relies on the in situ covalent bond formation between a benzylguanine-modified guide RNA (BG-gRNA) and a SNAP-tagged deaminase (SNAP-ADAR). Once the gRNA-deaminase conjugate is formed, it enables specific adenosine-to-inosine (A-to-I) substitutions in target RNAs. Since inosine is biochemically interpreted as guanosine by the cellular machinery, site-directed A-to-I editing provides the possibility to manipulate RNA and protein function. In this PhD project, it was aimed at elucidating the potential of the SNAP-ADAR approach for future applications. Therefore, the performance of the editing system in mammalian cells was comprehensively characterized. It could be shown that efficient site-directed RNA editing with SNAP-ADAR enzymes in cell culture requires the chemical modification of the BG-gRNA. A strong performance in the editing of endogenous transcripts was demonstrated in engineered cell lines stably expressing SNAP-ADAR enzymes. Editing yields up to 90% were achieved and remained stable even when several transcripts or multiple sites on a single transcript were concurrently targeted. Maximum editing was reached after 3 hours of BG-gRNA transfection and stayed unchanged for several days. Additionally, low concentrations (≥ 1.25 pmol/96-well) of the BG-gRNA were sufficient to obtain highest editing levels. The SNAP-ADAR approach holds great promise for the recoding of many functionally important amino acid residues as 11 out of the 16 adenosine-containing 5’-NAN triplets were editable between 50% and 90%. First evidence was provided that the editing system might be a valuable tool for the correction of disease-causing mutations. Moreover, the possibility of manipulating entire signaling networks was highlighted by the efficient and concurrent editing of two disease-relevant transcripts, KRAS and STAT1. Photo-controlled A-to-I editing was applied to direct protein localization within the cell by introducing alternative start or stop codons which allowed the expression of signals for nuclear and membrane translocation. NGS-based analysis revealed that wild-type SNAP-ADAR enzymes are highly precise editing machines. Their more active versions (SNAP-ADARQ enzymes) produced some off-target edits into the transcriptome, but the observed off-target activity appeared to be reducible by lowering the SNAP-ADAR protein amounts without great inhibition of the on-target editing. Nevertheless, these enzymes were one order more precise than editing machines applied by competing approaches. The chemical modification of the BG-gRNA was shown to suppress the off-target editing within a duplex formed by the BG-gRNA and the target RNA. The SNAP-ADAR approach outcompetes all well-characterized approaches for site-directed RNA editing due the best balance between efficiency and specificity

Effect of liquid viscosity on performance of wire mesh entrainment separators

The knitted wire mesh entrainment separator, commonly referred to as a demister*, is in common use to separate entrained liquid from vapor streams. However, little quantitative information is available concerning the effect of physical properties of the entrained liquid on allowable gas velocities. Studies were made on two demister styles to determine the effect of liquid viscosity on demister performance, Liquid viscosities from 0.9 to 12 centipoise were investigated using water, glycerine-water mixtures and heavy no. 2 fuel oil as the test liquids. Air was used as the gas medium in all cases. Regression analysis of the test data indicates that by increasing viscosity from 3.9 to 12 centipoise the allowable vapor velocity is decreased by only 10%. The effects of other liquid and demister properties on flooding velocity can be approximated by the following proposed equation: VFLOOD = 5.45 (ρl)0.47 (γ)0.20/((α/ε3)0.30 μl)0.036 (GL)0.11) * Registered trademark of Otto York Company

Nuclear magnetic resonance spectroscopy: Abnormal splitting of ethyl groups due to molecular asymmetry

Nuclear magnetic resonance (n.m.r.) spectroscopy provides an excellent means for qualitative identification of ethyl groups by use of the familiar three-four pattern of spin-spin splitting (1). It has been observed previously (2) that the methylene protons of systems of the type R-CH2-CR1R2R3 (where R1 can be the same as R or different) may be magnetically nonequivalent and display AB rather than A2-type spectra (3). We now wish to report several examples of this type of behavior with ethyl groups, particularly ethoxy groups, knowledge of which could be important to anyone using n.m.r. for organic qualitative analysis

Upper Extremity Biomechanical Model for Evaluation of Pediatric Joint Demands during Wheelchair Mobility

Current methods for evaluating upper extremity (UE) dynamics during pediatric wheelchair use are limited. We propose a new model to characterize UE joint kinematics and kinetics during pediatric wheelchair mobility. The bilateral model is comprised of the thorax, clavicle, scapula, upper arm, forearm, and hand segments. The modeled joints include: sternoclavicular, acromioclavicular, glenohumeral, elbow and wrist. The model is complete and is currently undergoing pilot studies for clinical application. Results may provide considerable quantitative insight into pediatric UE joint dynamics to improve wheelchair prescription, training and long term care of children with orthopaedic disabilities

Surface effects in the crystallization process of elastic flexible polymers

Investigating thermodynamic properties of liquid-solid transitions of flexible homopolymers with elastic bonds by means of multicanonical Monte Carlo simulations, we find crystalline conformations that resemble ground-state structures of Lennard-Jones clusters. This allows us to set up a structural classification scheme for finite-length flexible polymers and their freezing mechanism in analogy to atomic cluster formation. Crystals of polymers with "magic length" turn out to be perfectly icosahedral

Ventilation Transport Trade Study for Future Space Suit Life Support Systems

A new and advanced portable life support system (PLSS) for space suit surface exploration will require a durable, compact, and energy efficient system to transport the ventilation stream through the space suit. Current space suits used by NASA circulate the ventilation stream via a ball-bearing supported centrifugal fan. As NASA enters the design phase for the next generation PLSS, it is necessary to evaluate available technologies to determine what improvements can be made in mass, volume, power, and reliability for a ventilation transport system. Several air movement devices already designed for commercial, military, and space applications are optimized in these areas and could be adapted for EVA use. This paper summarizes the efforts to identify and compare the latest fan and bearing technologies to determine candidates for the next generation PLSS

IgG in the control of FcεRI activation: a battle on multiple fronts.

The rising global incidence of IgE-mediated allergic reactions poses a significant challenge to the quality of life of affected individuals and to healthcare systems, with current treatments being limited in effectiveness, safety, and disease-modifying capabilities. IgE acts by sensitizing the high-affinity IgE receptor FcεRI expressed by mast cells and basophils, tuning these cells for inflammatory degranulation in response to future allergen encounters. In recent years, IgG has emerged as an essential negative regulator of IgE-dependent allergic inflammation. Mechanistically, studies have proposed different pathways by which IgG can interfere with the activation of IgE-mediated inflammation. Here, we briefly summarize the major proposed mechanisms of action by which IgG controls the IgE-FcεRI inflammatory axis and how those mechanisms are currently applied as therapeutic interventions for IgE-mediated inflammation

State of the Work: Tackling the Tough Challenges to Advancing Diversity, Equity, and Inclusion

D5?s 2014 State of the Work highlights new tools and resources, recently completed research, and new organizational approaches that can help foundations and philanthropic organizations take action, achieve their goals regarding DEI, and advance the common good. The 2014 State of the Work also features perspectives and analysis of three thought leaders on DEI issues and their charge to their peers in philanthropy: Ange-Marie Hancock, Associate Professor of Political Science and Gender Studies at the University of Southern California; Todd Vogel, the Managing Director of Loom; and Gerri Spilka, Founding Director of the OMG Center for Collaborative Learning. The new report catalogs the progress D5 has made in laying the groundwork for a growing movement including several long-term projects that came to fruition in the past year:An Analysis of Policies, Practices, and Programs for Advancing DEI. In fall 2013, D5 released a comprehensive scan and analysis of scores of written and web-based resources.Deployment of a Regional Strategy. Recognizing that most philanthropy occurs on the local level, D5 complemented its national work by implementing a regional strategy, which targets DEI resources and support to pre-existing and emerging local networks in select regions of the country.Insights on Diversity: Three Commissioned Research Projects. D5 commissioned a series of research projects to explore: 1) The role of leadership in advancing DEI; 2) Career pathways for people of color in philanthropy; 3) The interactive factors between philanthropy and nonprofit organizations