Implementation of a learning healthcare system for sickle cell disease.

OBJECTIVE: Using sickle cell disease (SCD) as a model, the objective of this study was to create a comprehensive learning healthcare system to support disease management and research. A multidisciplinary team developed a SCD clinical data dictionary to standardize bedside data entry and inform a scalable environment capable of converting complex electronic healthcare records (EHRs) into knowledge accessible in real time. MATERIALS AND METHODS: Clinicians expert in SCD care developed a data dictionary to describe important SCD-associated health maintenance and adverse events. The SCD data dictionary was deployed in the EHR using EPIC SmartForms, an efficient bedside data entry tool. Additional data elements were extracted from the EHR database (Clarity) using Pentaho Data Integration and stored in a data analytics database (SQL). A custom application, the Sickle Cell Knowledgebase, was developed to improve data analysis and visualization. Utilization, accuracy, and completeness of data entry were assessed. RESULTS: The SCD Knowledgebase facilitates generation of patient-level and aggregate data visualization, driving the translation of data into knowledge that can impact care. A single patient can be selected to monitor health maintenance, comorbidities, adverse event frequency and severity, and medication dosing/adherence. CONCLUSIONS: Disease-specific data dictionaries used at the bedside will ultimately increase the meaningful use of EHR datasets to drive consistent clinical data entry, improve data accuracy, and support analytics that will facilitate quality improvement and research.

Experimental evolution of adenylate kinase reveals contrasting strategies toward protein thermostability.

Success in evolution depends critically upon the ability of organisms to adapt, a property that is also true for the proteins that contribute to the fitness of an organism. Successful protein evolution is enhanced by mutational pathways that generate a wide range of physicochemical mechanisms to adaptation. In an earlier study, we used a weak-link method to favor changes to an essential but maladapted protein, adenylate kinase (AK), within a microbial population. Six AK mutants (a single mutant followed by five double mutants) had success within the population, revealing a diverse range of adaptive strategies that included changes in nonpolar packing, protein folding dynamics, and formation of new hydrogen bonds and electrostatic networks. The first mutation, AK(BSUB) Q199R, was essential in defining the structural context that facilitated subsequent mutations as revealed by a considerable mutational epistasis and, in one case, a very strong dependence upon the order of mutations. Namely, whereas the single mutation AK(BSUB) G213E decreases protein stability by >25 degrees C, the same mutation in the background of AK(BSUB) Q199R increases stability by 3.4 degrees C, demonstrating that the order of mutations can play a critical role in favoring particular molecular pathways to adaptation. In turn, protein folding kinetics shows that four of the five AK(BSUB) double mutants utilize a strategy in which an increase in the folding rate accompanied by a decrease in the unfolding rate results in additional stability. However, one mutant exhibited a dramatic increase in the folding relative to a modest increase in the unfolding rate, suggesting a different adaptive strategy for thermostability. In all cases, an increase in the folding rates for the double mutants appears to be the preferred mechanism in conferring additional stability and may be an important aspect of protein evolution. The range of overlapping as well as contrasting strategies for success illustrates both the power and subtlety of adaptation at even the smallest unit of change, a single amino acid.

Human UP1 as a model for understanding purine recognition in the family of proteins containing the RNA recognition motif (RRM).

Heterogeneous ribonucleoprotein A1 (hnRNP A1) is a prototype for the family of eukaryotic RNA processing proteins containing the common RNA recognition motif (RRM). The region consisting of residues 1-195 of hnRNP A1 is referred to as UP1. This region has two RRMs and has a high affinity for both single-stranded RNA and the human telomeric repeat sequence d(TTAGGG)(n). We have used UP1's novel DNA binding to investigate how RRMs bind nucleic acid bases through their highly conserved RNP consensus sequences. Nine complexes of UP1 bound to modified telomeric repeats were investigated using equilibrium fluorescence binding and X-ray crystallography. In two of the complexes, alteration of a guanine to either 2-aminopurine or nebularine resulted in an increase in K(d) from 88nM to 209nM and 316nM, respectively. The loss of these orienting interactions between UP1 and the substituted base allows it to flip between syn and anti conformations. Substitution of the same base with 7-deaza-guanine preserves the O6/N1 contacts but still increases the K(d) to 296nM and suggests that it is not simply the loss of affinity that gives rise to the base mobility, but also the stereochemistry of the specific contact to O6. Although these studies provide details of UP1 interactions to nucleic acids, three general observations about RRMs are also evident: (1) as suggested by informatic studies, main-chain to base hydrogen bonding makes up an important aspect of ligand recognition (2) steric clashes generated by modification of a hydrogen bond donor-acceptor pair to a donor-donor pair are poorly tolerated and (3) a conserved lysine position proximal to RNP-2 (K(106)-IFVGGI) orients the purine to allow stereochemical discrimination between adenine and guanine based on the 6-position. This single interaction is well-conserved in known RRM structures and appears to be a broad indicator for purine preference in the larger family of RRM proteins.

Structure-based incorporation of 6-methyl-8-(2-deoxy-beta-ribofuranosyl)isoxanthopteridine into the human telomeric repeat DNA as a probe for UP1 binding and destabilization of G-tetrad structures.

Heterogeneous ribonucleoprotein A1 (hnRNP A1) is an abundant nuclear protein that participates in RNA processing, alternative splicing, and chromosome maintenance. hnRNP A1 can be proteolyzed to unwinding protein (UP1), a 22.1-kDa protein that retains a high affinity for purine-rich single-stranded nucleic acids, including the human telomeric repeat (hTR) d(TTAGGG)n. Using the structure of UP1 bound to hTR as a guide, we have incorporated the fluorescent guanine analog 6-MI at one of two positions within the DNA to facilitate binding studies. One is where 6-MI remains stacked with an adjacent purine, and another is where it becomes fully unstacked upon UP1 binding. The structures of both modified oligonucleotides complexed to UP1 were determined by x-ray crystallography to validate the efficacy of our design, and 6-MI has proven to be an excellent reporter molecule for single-stranded nucleic acid interactions in positions where there is a change in stacking environment upon complex formation. We have shown that UP1 affinity for d(TTAGGG)2 is approximately 5 nm at 100 mm NaCl, pH 6.0, and our binding studies with d(TTAGG(6-MI)TTAGGG) show that binding is only modestly sensitive to salt and pH. UP1 also has a potent G-tetrad destabilizing activity that reduces the Tm of the hTR sequence d(TAGGGT)4 from 67.0 degrees C to 36.1 degrees C at physiological conditions (150 mm KCl, pH 7.0). Consistent with the structures determined by x-ray crystallography, UP1 is able to bind the hTR sequence in solution as a dimer and supports a model for hnRNP A1 binding to nucleic acids in arrays that may make a contiguous set of anti-parallel single-stranded nucleic acid binding clefts. These data suggest that seemingly disparate roles for hnRNP A1 in alternative splice site selection, RNA processing, RNA transport, and chromosome maintenance reflect its ability to bind a purine-rich consensus sequence (nYAGGn) and destabilize potentially deleterious G-tetrad structures.