Structural Requirements for Ribosome-Dependent GTPase Activity and Binding

The two billion years of evolution since the divergence of prokaryotes and eukaryotes has left earth with very few molecules conserved across these two domains of life. One such molecule, the ribosome, is an enormous ribonucleoprotein responsible for translation, the process of converting the information contained within an organism’s genetic code into functional proteins. Translation is facilitated by a number of other proteins, termed translation factors, required to catalyze the synthesis of these proteins. A large number of antibiotics prescribed today target either the ribosome or translation factors, and with increasing antibiotic resistance being found in infectious bacteria there is a greater need for understanding the ribosome and its associated molecules. While knowledge of the ribosome has increased considerably over the past two decades there are still many unknowns surrounding its transition states, how it interacts with translation factors, and even the role some of these translation factors play in the cell. A more thorough understanding of the interactions between ribosomes and translation factors will lead to the generation of more diverse and possibly more potent antibiotics for the treatment of bacterial infections. The goals of this work were threefold. First, based on the work of previous Spiegel lab graduate student Justin Walter, we aimed to further characterize the role of the L12 ribosomal protein in activation of several translation factors that v utilize the hydrolysis of guanosine 5’-triphosphate (GTP), called GTPases, to exert their function. Walter had shown that removal of L12 led to decreased GTP hydrolysis by these proteins, but was unable to ensure L12 had been completely removed from the ribosomes. Here it is shown that when L12 is completely absent from functional prokaryotic ribosomes the GTPase activity of three translation factors, elongation factor G (EF-G), release factor 3 (RF3), and initiation factor 2 (IF2) all unequivocally cease, showing no activity beyond that of uncatalyzed GTP hydrolysis. A fourth translational GTPase, leader peptidase A (LepA), exhibited a different response, with activity dropping by effectively 50% upon the removal of L12. Reconstitution of these depleted ribosomes with externally purified L12 caused an unambiguous return to full activity for all investigated GTPases. A second ambition of this work was to analyze the role of the L12 ribosomal protein in binding of translation factors. GTPase binding assays through ultracentrifugation demonstrated that absolute removal of L12 led to a nearly complete abrogation of binding between 70S ribosomes and EF-G, IF2, and RF3. LepA exhibited diminished binding in the presence of L12 deficient ribosomes, but maintained a level significantly above baseline. To further assess the effect of L12 depletion on binding, BioLayer Interferometry was utilized to quantitatively measure the binding affinity between EF-G and 70S or 70SΔL12. EF-G and 70S interactions fell within previously established KD values, averaging ~160 nM. Preincubation of EF-G with 70SΔL12 maintained this affinity, suggesting that little vi to no EF-G associates with depleted ribosomes, while preincubation of EF-G with intact 70S ribosomes caused a \u3e 10,000 fold increase in the KD, indicating EF-G has a strong association with 70S ribosomes when L12 is present. The final objective herein was to determine the roles of domains 4 and 5 and subdomain G’ of EF-G in the hydrolysis of GTP. EF-GΔ5 and EF-GΔ4,5 were previously produced in the Spiegel lab. Here it is shown that EF-GΔ4 and EF-GΔG’ are both expressed in the soluble fraction of E. coli cells and are readily isolated. The GTPase activity of each mutant relative to full length EF-G was calculated. EF-GΔ4 and EF-GΔ4,5 exhibited an activity of roughly 65% of wild type EF-G, suggesting the loss of the 4 domain confers the same disadvantage as the loss of the 4 and 5 domains. Meanwhile, EF-GΔ5 maintained 85% activity, showing the loss of the 5 domain is less detrimental to GTPase activity than either the Δ4 or Δ4,5 mutants. EF-GΔG’ confers a loss of around 90% activity compared to EF-G, suggestive of a crucial role of this domain in EF-G activity or binding, despite being absent in other homologous translational GTPases

An examination of the effects of self-regulatory focus on the perception of the media richness: the case of email

Communication is a key element in organizations’ business success. The media richness theory and the channel expansion theory are two of the most influential theories regarding the selection and use of communication media in organizations; however, literature has focused little on the effects of self-regulation by managers and employees in these theories. To analyze these topics, this study develops an empirical investigation by gathering data from 600 managers and employees using a questionnaire. The results suggest that the perception of media richness is positively affected when the individual shows a promotion focus or strategy.Peer ReviewedPostprint (author’s final draft

The continuum of spreading depolarizations in acute cortical lesion development: Examining Leao's legacy

A modern understanding of how cerebral cortical lesions develop after acute brain injury is based on Aristides Leao's historic discoveries of spreading depression and asphyxial/anoxic depolarization. Treated as separate entities for decades, we now appreciate that these events define a continuum of spreading mass depolarizations, a concept that is central to understanding their pathologic effects. Within minutes of acute severe ischemia, the onset of persistent depolarization triggers the breakdown of ion homeostasis and development of cytotoxic edema. These persistent changes are diagnosed as diffusion restriction in magnetic resonance imaging and define the ischemic core. In delayed lesion growth, transient spreading depolarizations arise spontaneously in the ischemic penumbra and induce further persistent depolarization and excitotoxic damage, progressively expanding the ischemic core. The causal role of these waves in lesion development has been proven by real-time monitoring of electrophysiology, blood flow, and cytotoxic edema. The spreading depolarization continuum further applies to other models of acute cortical lesions, suggesting that it is a universal principle of cortical lesion development. These pathophysiologic concepts establish a working hypothesis for translation to human disease, where complex patterns of depolarizations are observed in acute brain injury and appear to mediate and signal ongoing secondary damage

Mononuclear cells modulate the activity of pancreatic stellate cells which in turn promote fibrosis and inflammation in chronic pancreatitis

Background: Interactions between mononuclear cells and activated pancreatic myofibroblasts (pancreatic stellate cells; PSC) may contribute to inflammation and fibrosis in chronic pancreatitis (CP). Methods: Markers of fibrosis and inflammation were concomitantly analysed by immunohistochemistry in chronic pancreatitis tissues. In vitro, PSC were stimulated with TNFalpha and LPS. Primary human blood mononuclear cells (PBMC) and PSC were cocultured, followed by analysis of cytokines and extracellular matrix (ECM) proteins. PBMC were derived from healthy donors and CP and septic shock patients. Results: In areas of mononuclear cell infiltration in chronic pancreatitis tissues, there was decreased immunoreactivity for collagen1 and fibronectin, in contrast to areas with sparse mononuclear cells, although PSC were detectable in both areas. LPS and TNFalpha induced collagen1 and fibronectin levels as well as the matrix degradation enzyme MMP-1. Coculture experiments with PSC and PBMC revealed increased fibronectin secretion induced by PBMC. In addition, donor and CP PBMC significantly induced an increase in IL-6, MCP-1 and TGFbeta levels under coculture conditions. Determination of the source of cytokines and ECM proteins by mRNA expression analysis confirmed PSC as major contributors of ECM production. The increase in cytokine expression was PBMC- and also PSC-derived. Conclusion: Mononuclear cells modulate the activity of pancreatic stellate cells, which may in turn promote fibrosis and inflammation

Crystal Structure Analysis Reveals Functional Flexibility in the Selenocysteine-Specific tRNA from Mouse

Selenocysteine tRNAs (tRNA(Sec)) exhibit a number of unique identity elements that are recognized specifically by proteins of the selenocysteine biosynthetic pathways and decoding machineries. Presently, these identity elements and the mechanisms by which they are interpreted by tRNA(Sec)-interacting factors are incompletely understood.We applied rational mutagenesis to obtain well diffracting crystals of murine tRNA(Sec). tRNA(Sec) lacking the single-stranded 3'-acceptor end ((ΔGCCA)RNA(Sec)) yielded a crystal structure at 2.0 Å resolution. The global structure of (ΔGCCA)RNA(Sec) resembles the structure of human tRNA(Sec) determined at 3.1 Å resolution. Structural comparisons revealed flexible regions in tRNA(Sec) used for induced fit binding to selenophosphate synthetase. Water molecules located in the present structure were involved in the stabilization of two alternative conformations of the anticodon stem-loop. Modeling of a 2'-O-methylated ribose at position U34 of the anticodon loop as found in a sub-population of tRNA(Sec)in vivo showed how this modification favors an anticodon loop conformation that is functional during decoding on the ribosome. Soaking of crystals in Mn(2+)-containing buffer revealed eight potential divalent metal ion binding sites but the located metal ions did not significantly stabilize specific structural features of tRNA(Sec).We provide the most highly resolved structure of a tRNA(Sec) molecule to date and assessed the influence of water molecules and metal ions on the molecule's conformation and dynamics. Our results suggest how conformational changes of tRNA(Sec) support its interaction with proteins

Nurse-patient interaction and communication: a systematic literature review

Aim: The purpose of this review is to describe the use and definitions of the concepts of nurse-patient interaction and nurse-patient communication in nursing literature. Furthermore, empirical findings of nurse-patient communication research will be presented, and applied theories will be shown. Method: An integrative literature search was executed. The total number of relevant citations found was 97. The search results were reviewed, and key points were extracted in a standardized form. Extracts were then qualitatively summarized according to relevant aspects and categories for the review. Results: The relation of interaction and communication is not clearly defined in nursing literature. Often the terms are used interchangeably or synonymously, and a clear theoretical definition is avoided or rather implicit. Symbolic interactionism and classic sender-receiver models were by far the most referred to models. Compared to the use of theories of adjacent sciences, the use of original nursing theories related to communication is rather infrequent. The articles that try to clarify the relation of both concepts see communication as a special or subtype of interaction. Conclusion: The included citations all conclude that communication skills can be learned to a certain degree. Involvement of patients and their role in communication often is neglected by authors. Considering the mutual nature of communication, patients’ share in conversation should be taken more into consideration than it has been until now. Nursing science has to integrate its own theories of nursing care with theories of communication and interaction from other scientific disciplines like sociology

A Population of Gamma-Ray Millisecond Pulsars Seen with the Fermi Large Area Telescope

Gamma-Ray Pulsar Bonanza Most of the pulsars we know about were detected through their radio emission; a few are known to pulse gamma rays but were first detected at other wavelengths (see the Perspective by Halpern ). Using the Fermi Gamma-Ray Space Telescope, Abdo et al. (p. 840 , published online 2 July; see the cover) report the detection of 16 previously unknown pulsars based on their gamma-ray emission alone. Thirteen of these coincide with previously unidentified gamma-ray sources, solving the 30-year-old mystery of their identities. Pulsars are fast-rotating neutron stars. With time they slow down and cease to radiate; however, if they are in a binary system, they can have their spin rates increased by mass transfer from their companion stars, starting a new life as millisecond pulsars. In another study, Abdo et al. (p. 845 ) report the detection of gamma-ray emission from the globular cluster 47 Tucanae, which is coming from an ensemble of millisecond pulsars in the cluster's core. The data imply that there are up to 60 millisecond pulsars in 47 Tucanae, twice as many as predicted by radio observations. In a further companion study, Abdo et al. (p. 848 , published online 2 July) searched Fermi Large Area Telescope data for pulsations from all known millisecond pulsars outside of stellar clusters, finding gamma-ray pulsations for eight of them. Their properties resemble those of other gamma-ray pulsars, suggesting that they share the same basic emission mechanism. Indeed, both sets of pulsars favor emission models in which the gamma rays are produced in the outer magnetosphere of the neutron star