Structural studies of the ribosome have benefited greatly from the use of organisms adapted to extreme environments. However, little is known about the mechanisms by which ribosomes or other ribonucleoprotein complexes have adapted to functioning under extreme conditions, and it is unclear to what degree mutant phenotypes of extremophiles will resemble those of their counterparts adapted to more moderate environments. It is conceivable that phenotypes of mutations affecting thermophilic ribosomes, for instance, will be influenced by structural adaptations specific to a thermophilic existence. This consideration is particularly important when using crystal structures of thermophilic ribosomes to interpret genetic results from nonextremophilic species. To address this issue, we have conducted a survey of spontaneously arising antibioticresistant mutants of the extremely thermophilic bacterium Thermus thermophilus, a species which has featured prominently in ribosome structural studies. We have accumulated over 20 single-base substitutions in T. thermophilus 16S and 23S rRNA, in the decoding site and in the peptidyltransferase active site of the ribosome. These mutations produce phenotypes that are largely identical to those of corresponding mutants of mesophilic organisms encompassing a broad phylogenetic range, suggesting that T. thermophilus may be an ideal model system for the study of ribosome structure and function.Members of the bacterial genus Thermus are extreme thermophiles first described by Brock and Freeze in 1969 (2) and have since been found in terrestrial and marine thermal environments throughout the world (60). Together with Deinococcus, Meiothermus, Marinithermus, Oceanithermus, and Vulcanithermus, they form a deeply branching phylum now known to be monophyletic (23,59). The close affiliation between Thermus and Deinococcus has been confirmed by complete genome sequences of Thermus thermophilus (30) and Deinococcus radiodurans (59). The finding of slightly thermophilic species related to Deinococcus, together with the thermophilic nature of the other genera of this phylum, suggests that thermophily is a primitive character of this clade (59).Recent advances in structural biology have produced a vast reservoir of high-resolution structural information regarding components of the protein synthetic machinery from members of the Deinococcus-Thermus phylum, including high-resolution crystal structures of the T. thermophilus 30S subunit (47, 61), medium-resolution structures of the entire T. thermophilus 70S ribosome (65), and high-resolution structures of the D. radiodurans 50S subunit (26). The value of these structures is magnified by their interpretation in light of several decades of genetics and biochemistry using ribosomes from mesophiles such as Escherichia coli. However, the ability to make such interpretations is potentially compromised by the absence of genetic and biochemical data obtained for ribosomes from members of the Deinococcus-Thermus phylum. It is assumed that nucleotide sequence c...

A mechanistic understanding of ribosome function demands knowledge of the conformational changes that occur during protein synthesis. One current model proposes a conformational switch in Helix 27 (H27) of 16S rRNA involved in the decoding of mRNA. This model was based on the behavior of mutations in the 912 region of H27 of Escherichia coli 16S rRNA, which were predicted to stabilize the helix in either of two alternative conformations. This interpretation was supported by evidence from both genetics and structural biochemistry. However, recently published X-ray crystallographic structures of the Thermus thermophilus 30S subunit at different stages of tRNA selection have raised doubts regarding the validity of this model. We have therefore revisited the model genetically by constructing a H27 quadruple mutation (C912G, C910G, G885C, and G887C), which would create multiple mismatches in the proposed alternative conformation without perturbing the native H27 conformation seen in the crystal structures. Inconsistent with the H27 switch model, cells containing pure populations of quadruple mutant ribosomes grow at essentially wild-type rates. The mutants used to construct the H27 switch model all carried A2058G in 23S rRNA and C1192U in 16S rRNA as selectable markers. The quadruple mutant carrying these additional marker mutations is deleterious, and we conclude that they have a synergistic effect when combined with other mutations and are not phenotypically silent. Their presence confounded the interpretation of the original mutant phenotypes and, in light of the viability of the quadruple mutant, we conclude that the genetic evidence no longer supports the model.

Defects in DNA mismatch repair predispose cells to the development of several types of malignant disease. The absence of Msh2 or Mlh1, two key molecules that mediate mismatch repair in eukaryotic cells, increases the frequency of mutation and also alters the response of some cells to apoptosis and cell cycle arrest. To understand the way these changes contribute to cancer predisposition, we examined the effects of defective mismatch repair on the multistep process of pre-B-cell transformation by Abelson murine leukemia virus. In this model, primary transformants undergo a prolonged apoptotic crisis followed by the emergence of fully transformed cell lines. The latter event is correlated to a loss of function of the p53 tumor suppressor protein and down-modulation of the p53 regulatory protein p19Arf. Analyses of primary transformants from Msh2 null mice and their wild-type littermates revealed that both types of cells undergo crisis. However, primary transformants from Msh2 null animals recover with accelerated kinetics, a phenomenon that is strongly correlated to the appearance of cells that have lost p53 function. Analysis of the kinetics with which p53 function is lost revealed that this change provides the dominant stimulus for emergence from crisis. Therefore, the absence of mismatch repair alters the molecular mechanisms involved in transformation by affecting a gene that controls apoptosis and cell cycle progression, rather than by affecting these processes directly.

Throughout evolution, emerging organisms survived by adapting existing biochemical processes to new reaction conditions. Simple protein enzymes balanced changes in structural stability with changes that permitted optimal catalysis by adjustments in both entropic and enthalpic contributions to the free energy of activation for the reaction. Study of adaptive mechanisms by large multicomponent enzymes such as the ribosome has been largely unexplored. Here we have determined the kinetic and thermodynamic parameters of peptidyltransferase in ribosomes from the extreme thermophile Thermus thermophilus. Activity of thermophilic enzymes can be assayed over a wide range of temperatures, enabling one to measure accurate catalytic rates and determine enthalpic and entropic contributions to the free energy of activation of the reaction. Differences in the reaction conditions used here and in published studies on mesophilic ribosomes prevent direct comparison, but our data on Thermus ribosomes suggest that these ribosomes have adapted to changing environments using the same strategies as simple protein enzymes, balancing stability and flexibility without loss of catalytic rate. This strategy must be a very ancient process, perhaps first used by primitive ribosomes in the RNA World.

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Background: Morbidity and mortality (M&M) conferences are essential components for resident education and provide a valuable tool to improve patient safety and quality of care. M&M conferences help identify important gaps in safety and reduce avoidable events in future patient care. Active methods to improve the utilization of M&M conferences have been shown to enhance their educational value for residents, faculty and multidisciplinary teams in healthcare institutions. Objective: The purpose of this study was to use a survey-based methodology to assess how morbidity and mortality conferences are conducted in residency programs, including characteristics such as frequency, involvement of personnel and the effects of COVID-19. Methods: From February to October 2021, a validated 19 question survey was electronically distributed to residency program directors in anesthesiology, emergency medicine and general surgery, after a search for email addresses in the ACGME database. The survey was created and hosted on Google Forms. Results: A total of 125 of 713 program directors (17.5%) responded to the survey. Eighty-three percent of respondent programs reported mandatory participation for residents, with residents providing most of the presentations. Case presentations utilized various formats including SBAR, adverse event analysis and root cause analysis as the most common modalities. Though most programs reported no change in frequency of M&M conferences due to COVID-19, most respondents reported a shift to a virtual or hybrid platform. Conclusion: M&M conferences are an important educational and quality improvement modality, and many residency directors changed practice to incorporate virtual platforms due to the COVID-19 pandemic to maintain uninterrupted educational sessions. Nonetheless, significant variation still exists in how these conferences are conducted between different institutions.

The increased demand for procedural sedation in the ambulatory setting has prompted the development of anesthetic agents that anesthesiologists and non-anesthesiologists could administer easily and safely. In this article, we discuss novel short-acting agents in development or in clinical trials that may serve as alternatives to current anesthetics for procedural sedation.

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