In vitro selected GUAA tetraloop-binding receptors with structural plasticity and evolvability towards natural RNA structural modules.

GNRA tetraloop-binding receptor interactions are key components in the macromolecular assembly of a variety of functional RNAs. In nature, there is an apparent bias for GAAA/11nt receptor and GYRA/helix interactions, with the former interaction being thermodynamically more stable than the latter. While past in vitro selections allowed isolation of novel GGAA and GUGA receptors, we report herein an in vitro selection that revealed several novel classes of specific GUAA receptors with binding affinities comparable to those from natural GAAA/11nt interactions. These GUAA receptors have structural homology with double-locked bulge RNA modules naturally occurring in ribosomal RNAs. They display mutational robustness that enables exploration of the sequence/phenotypic space associated to GNRA/receptor interactions through epistasis. Their thermodynamic self-assembly fitness landscape is characterized by a rugged neutral network with possible evolutionary trajectories toward natural GNRA/receptor interactions. High throughput sequencing analysis revealed synergetic mutations located away from the tertiary interactions that positively contribute to assembly fitness. Our study suggests that the repertoire of GNRA/receptor interactions is much larger than initially thought from the analysis of natural stable RNA molecules and also provides clues for their evolution towards natural GNRA/receptors.

Deducing putative ancestral forms of GNRA/receptor interactions from the ribosome.

Stable RNAs rely on a vast repertoire of long-range interactions to assist in the folding of complex cellular machineries such as the ribosome. The universally conserved L39/H89 interaction is a long-range GNRA-like/receptor interaction localized in proximity to the peptidyl transferase center of the large subunit of the ribosome. Because of its central location, L39/H89 likely originated at an early evolutionary stage of the ribosome and played a significant role in its early function. However, L39/H89 self-assembly is impaired outside the ribosomal context. Herein, we demonstrate that structural modularity principles can be used to re-engineer L39/H89 to self-assemble in vitro. The new versions of L39/H89 improve affinity and loop selectivity by several orders of magnitude and retain the structural and functional features of their natural counterparts. These versions of L39/H89 are proposed to be ancestral forms of L39/H89 that were capable of assembling and folding independently from proteins and post-transcriptional modifications. This work demonstrates that novel RNA modules can be rationally designed by taking advantage of the modular syntax of RNA. It offers the prospect of creating new biochemical models of the ancestral ribosome and increases the tool kit for RNA nanotechnology and synthetic biology.

Developing a Curriculum for Change: Water and Environmental Health Literacy in a Native American Community.

BACKGROUND: Knowing about risks of water contamination is the first step in making informed choices to protect our health and environment. Researchers were challenged with sharing water quality research this information with local communities. OBJECTIVES: The purpose of this article is to describe the formative evaluation used to develop and implement an Environmental Health Literacy (EHL) summer camp and afterschool water curriculum for Native American (NA) children in the fourth through sixth grades. METHODS: Community and university scientists, elders, and educators came together and co-developed a summer camp and afterschool program for local youth to address the issues of water and its importance to the tribal community. LESSONS LEARNED: Research partners must continually balance research needs with relationships and service to the community. The health literacy framework used to develop our curriculum also complemented our community-based participatory research (CBPR) approach and may benefit other partnerships. CBPR helped to build trust and culturally center the intervention. CONCLUSIONS: Project partners built on the mutual commitment to "do what we say we will do" within the community context. Using the CBPR approach provided a strong framework for the collaboration necessary for this project. Trust relationships were key to the successes experienced during the development, implementation, and multiple revisions of this intervention.

Attenuation of loop-receptor interactions with pseudoknot formation.

RNA tetraloops can recognize receptors to mediate long-range interactions in stable natural RNAs. In vitro selected GNRA tetraloop/receptor interactions are usually more 'G/C-rich' than their 'A/U-rich' natural counterparts. They are not as widespread in nature despite comparable biophysical and chemical properties. Moreover, while AA, AC and GU dinucleotide platforms occur in natural GAAA/11 nt receptors, the AA platform is somewhat preferred to the others. The apparent preference for 'A/U-rich' GNRA/receptor interactions in nature might stem from an evolutionary adaptation to avoid folding traps at the level of the larger molecular context. To provide evidences in favor of this hypothesis, several riboswitches based on natural and artificial GNRA receptors were investigated in vitro for their ability to prevent inter-molecular GNRA/receptor interactions by trapping the receptor sequence into an alternative intra-molecular pseudoknot. Extent of attenuation determined by native gel-shift assays and co-transcriptional assembly is correlated to the G/C content of the GNRA receptor. Our results shed light on the structural evolution of natural long-range interactions and provide design principles for RNA-based attenuator devices to be used in synthetic biology and RNA nanobiotechnology.

Downward causation by information control in micro-organisms.

The concepts of functional equivalence classes and information control in living systems are useful to characterize downward (or top-down) causation by feedback information control in synthetic biology. Herein, we re-analyse published experiments of microbiology and synthetic biology that demonstrate the existence of several classes of functional equivalence in microbial organisms. Classes of functional equivalence from the bacterial operating system, which processes and controls the information encoded in the genome, can readily be interpreted as strong evidence, if not demonstration, of top-down causation (TDC) by information control. The proposed biological framework reveals how this type of causality is put in action in the cellular operating system. Considerations on TDC by information control and adaptive selection can be useful for synthetic biology by delineating the irreducible set of properties that characterizes living systems. Through a 'retro-synthetic' biology approach, these considerations could contribute to identifying the constraints behind the emergence of molecular complexity during the evolution of an ancient RNA/peptide world into a modern DNA/RNA/protein world. In conclusion, we propose TDCs by information control and adaptive selection as the two types of downward causality absolutely necessary for life.

Spirituality in medical school curricula: findings from a national survey.

OBJECTIVE: No systematic information exists on what U.S. medical schools are teaching on spirituality and health or on the attitudes of faculty toward inclusion of this subject in the medical curriculum. We systematically surveyed U.S. medical school deans and assessed both attitudes about and the extent to which spirituality is addressed in medical school curricula. METHODS: The responses to a questionnaire were solicited from deans representing 122 U.S. medical schools accredited by the Liaison Committee for Medical Education. Completed surveys were received from 85% (n = 104), with 94% (n = 115) responding to the primary question. Outcomes were proportion of medical schools with curricular content on spirituality and attitudes of deans toward such material. RESULTS: Ninety percent (range 84%-90%) of medical schools have courses or content on spirituality and health (S&H), 73% with content in required courses addressing other topics and 7% with a required course dedicated to S&H. Although over 90% indicate that patients emphasize spirituality in their coping and health care, only 39% say that including S&H is important. When asked if their institution needs more S&H curricular content, 43% indicated they did; however, even if funding and training support were available, only 25% would open additional curricular time. National policy statements, established competencies, or methods to evaluate student competencies in S&H were generally considered unimportant. CONCLUSIONS: Most U.S. medical schools have curricular content on S&H, although this varies greatly in scope. Despite acknowledging its importance to patients, the majority of deans are uncertain about including spirituality and do not think more content is needed.