Minimal Increments of Hydrophobic Collapse within the N-Terminus of the Neuropeptide Galanin.

The neuropeptide galanin has a 35-year history as an intriguing target in drug design owing to its implication as a potential anticonvulsant and neuronal trophic factor among many other therapeutically interesting functions including analgesia and mood alteration. In this study, we report the structural characterization of three synthetic fragments of the galanin N-terminus in buffered aqueous solution: hGal(2-12)KK, hGal(1-12)KK, and hGal(1-17)KK. High-field two-dimensional 1H-1H nuclear magnetic resonance (NMR) data were acquired for these fragments and used to derive distance restraints. We further utilized modified hydrogen bonding and dihedral restraints to reflect chemical shift patterns in the data, which revealed the signature of a weakly folded helix. Together, these sets of restraints were used to generate NMR structures of all three fragments, which depict a core of hydrophobic residues that cluster together regardless of the presence of a helical structure, and correspond to residues in the N-terminus of galanin that have been previously shown to be critical for binding its receptors. The helical structure only appears following the inclusion of Gly(1) in the sequence, and at longer sequence lengths, unlike many other peptides, the helix does not propagate. Rather, a few turns of poorly ordered helix appear to be a secondary consequence of clusters of hydrophobic sidechains that are conserved across all of the peptides in this study; the helices themselves appear ordered as a consequence of this clustering, and these clusters compare directly to those observed recently to make contacts between galanin and two of its receptor subtypes. Collapsed hydrophobic residues therefore organize and compose the functional core of human galanin and raise interesting questions about the nature of the conformational order in ligands that bind cell surface receptors.

The neuropeptide galanin adopts an irregular secondary structure.

Human galanin is a 30-residue neuropeptide targeted for development of analgesics, antidepressants, and anticonvulsants. While previous work from our group and others has already produced significant insights into galanin's N-terminal region, no extant structures of galanin in databases include its full-length sequence and the function of its C-terminus remains ambiguous. We report the NMR solution structure of full-length human galanin C-terminal amide, determined from 2D 1H-1H COSY, TOCSY, and ROESY NMR data. Galanin adopts an irregular helical structure across its N-terminus, likely the average of several coiling states. We present the NMR structure of a peptide encompassing the C-terminus of galanin as a stand-alone fragment. The C-terminus of full-length galanin appears to indirectly assist the intramolecular association of hydrophobic sidechains within its N-terminus, remotely rigidifying their position when compared to previously studied N-terminal galanin fragments. By contrast, there is flexibility in the C-terminus of galanin, characterized by two i to i + 2 hydrogen-bonded turns within an otherwise dynamic backbone. The C-terminal portion of the peptide renders it soluble, and plays a hitherto undescribed biophysical role in pre-organizing the galanin receptor binding epitope. We speculate that hydrophilic microdomains of signaling peptides, hormones, and perhaps intrinsically disordered proteins may also function similarly.

Sequential and Environmental Dependence of Conformation in a Small Opioid Peptide.

We report the structural characterization of the µ-selective endogenous opioid endomorphin-1 (EM-1) via an array of nuclear magnetic resonance experiments in both aqueous conditions and, for the first time, in isotropic lipid bicelles, which mimic its endogenous environment. Consistent with computationally derived hypotheses, EM-1 is found to significantly populate a compact, turn-like structure in aqueous solution. This structure is only present as a minor species when the peptide is subjected to a lipid environment, in which the presented NMR data suggests that the major conformer of EM-1 lacks internal hydrogen bonds. The interaction of EM-1 with lipid bilayers is characterized by both tryptophan fluorescence and two-dimensional diffusion ordered NMR spectroscopy; these experiments suggest that the interaction with the surface of phospholipid bilayers, operating as a change in bulk solvation, is responsible for the observed conformational rearrangement in EM-1.

Site-Selective Reactions with Peptide-Based Catalysts.

The problem of catalyst-controlled site-selectivity can potentially require a catalyst to overcome energetic barriers larger than those associated with enantioselective reactions. This challenge is a signature of substrates that present reactive sites that are not of equivalent reactivity. Herein we present a narrative of our laboratory's efforts to overcome this challenge using peptide-based catalysts. We highlight the interplay between understanding the inherent reactivity preferences of a given target molecule and the development of catalysts that can overcome intrinsic preferences embedded within a substrate.

A Synergistic Combinatorial and Chiroptical Study of Peptide Catalysts for Asymmetric Baeyer-Villiger Oxidation.

We report an approach to the asymmetric Baeyer-Villiger oxidation utilizing bioinformatics-inspired combinatorial screening for catalyst discovery. Scaled-up validation of our on-bead efforts with a circular dichroism-based assay of alcohols derived from the products of solution-phase reactions established the absolute configuration of lactone products; this assay proved equivalent to HPLC in its ability to evaluate catalyst performance, but was far superior in its speed of analysis. Further solution-phase screening of a focused library suggested a mode of asymmetric induction that draws distinct parallels with the mechanism of Baeyer-Villiger monooxygenases.

A γ-amino acid that favors 12/10-helical secondary structure in α/γ-peptides.

H-bonded helices in conventional peptides (containing exclusively homochiral α-amino acid residues) feature a uniform H-bonding directionality, N-terminal side C═O to C-terminal side NH. In contrast, heterochiral α-peptides can form helices in which the H-bond directionality alternates along the backbone because neighboring amide groups are oriented in opposite directions. Alternating H-bond directions are seen also in helices formed by unnatural peptidic backbones, e.g., those containing ß- or γ-amino acid residues. In the present study, we used NMR spectroscopy and crystallography to evaluate the conformational preferences of the novel γ-amino acid (1R,2R,3S)-2-(1-aminopropyl)-cyclohexanecarboxylic acid (APCH), which is constrained by a six-membered ring across its Cα-Cß bond. These studies were made possible by the development of a stereoselective synthesis of N-protected APCH. APCH strongly enforces the α/γ-peptide 12/10-helical secondary structure, which features alternating H-bond directionality. Thus, APCH residues appear to have a conformational propensity distinct from those of other cyclically constrained γ-amino acid residues.

Evaluation of a cyclopentane-based γ-amino acid for the ability to promote α/γ-peptide secondary structure.

We report the asymmetric synthesis of the γ-amino acid (1R,2R)-2-aminomethyl-1-cyclopentane carboxylic acid (AMCP) and an evaluation of this residue's potential to promote secondary structure in α/γ-peptides. Simulated annealing calculations using NMR-derived distance restraints obtained for α/γ-peptides in chloroform reveal that AMCP-containing oligomers are conformationally flexible. However, additional evidence suggests that an internally hydrogen-bonded helical conformation is partially populated in solution. From these data, we propose characteristic NOE patterns for the formation of the α/γ-peptide 12/10-helix and discuss the apparent conformational frustration of AMCP-containing oligomers.

Differential diagnosis generators: an evaluation of currently available computer programs.

BACKGROUND: Differential diagnosis (DDX) generators are computer programs that generate a DDX based on various clinical data. OBJECTIVE: We identified evaluation criteria through consensus, applied these criteria to describe the features of DDX generators, and tested performance using cases from the New England Journal of Medicine (NEJM©) and the Medical Knowledge Self Assessment Program (MKSAP©). METHODS: We first identified evaluation criteria by consensus. Then we performed Google® and Pubmed searches to identify DDX generators. To be included, DDX generators had to do the following: generate a list of potential diagnoses rather than text or article references; rank or indicate critical diagnoses that need to be considered or eliminated; accept at least two signs, symptoms or disease characteristics; provide the ability to compare the clinical presentations of diagnoses; and provide diagnoses in general medicine. The evaluation criteria were then applied to the included DDX generators. Lastly, the performance of the DDX generators was tested with findings from 20 test cases. Each case performance was scored one through five, with a score of five indicating presence of the exact diagnosis. Mean scores and confidence intervals were calculated. KEY RESULTS: Twenty three programs were initially identified and four met the inclusion criteria. These four programs were evaluated using the consensus criteria, which included the following: input method; mobile access; filtering and refinement; lab values, medications, and geography as diagnostic factors; evidence based medicine (EBM) content; references; and drug information content source. The mean scores (95% Confidence Interval) from performance testing on a five-point scale were Isabel© 3.45 (2.53, 4.37), DxPlain® 3.45 (2.63-4.27), Diagnosis Pro® 2.65 (1.75-3.55) and PEPID™ 1.70 (0.71-2.69). The number of exact matches paralleled the mean score finding. CONCLUSIONS: Consensus criteria for DDX generator evaluation were developed. Application of these criteria as well as performance testing supports the use of DxPlain® and Isabel© over the other currently available DDX generators.

Synthesis and crystal structure of (2S,4aR,8aR)-6-oxo-2,4a,6,8a-tetra-hydro-pyrano[3,2-b]pyran-2-carboxamide.

The pyran-opyran amide (2S,4aR,8aR)-6-oxo-2,4a,6,8a-tetra-hydro-pyrano[3,2-b]pyran-2-carboxamide, C9H9NO4, 3, was prepared by a chemoselective hydration of the corresponding nitrile, 2, using a heterogeneous catalytic method based on copper(II) supported on mol-ecular sieves, in the presence of acetaldoxime. Compound 3 belongs to a new class of pyran-opyrans that possess anti-bacterial and phytotoxic activity. Crystallographic analysis of 3 shows a bent structure for the cis-fused bicyclic pyran-opyran, similar to nitrile 2. Evidence of an intra-molecular hydrogen bond involving the amide group and ring oxygen was not observed; however, two separate inter-molecular hydrogen-bonding inter-actions were observed between the amide hydrogen atoms and adjacent carbonyl oxygen atoms along the b- and a-axis directions. The latter inter-action may also be supported by an inter-molecular C-H⋯O hydrogen bond. The lattice is filled out by close-packed layers of this hydrogen-bonded network along the c-axis direction, related from one to the next by a 21 screw axis.

An alpha/beta-peptide helix bundle with a pure beta3-amino acid core and a distinctive quaternary structure.

Helix bundles are among the most widely studied tertiary and quaternary structural motifs in proteins. Here we present the crystal structure of an alpha/beta-peptide foldamer that adopts a tetrameric helix-bundle quaternary structure with a hydrophobic core composed solely of beta-amino acids. The structure displays features that are unprecedented among all known helix bundles composed of either alpha-peptides or peptidic foldamers. The tetramer is characterized by an asymmetry of interaction between neighboring helices, and the side-chain packing within the hydrophobic core differs fundamentally from the knobs-into-holes arrangement typical of most helix bundles.

Competencies for improving diagnosis: an interprofessional framework for education and training in health care.

Background Given an unacceptably high incidence of diagnostic errors, we sought to identify the key competencies that should be considered for inclusion in health professions education programs to improve the quality and safety of diagnosis in clinical practice. Methods An interprofessional group reviewed existing competency expectations for multiple health professions, and conducted a search that explored quality, safety, and competency in diagnosis. An iterative series of group discussions and concept prioritization was used to derive a final set of competencies. Results Twelve competencies were identified: Six of these are individual competencies: The first four (#1-#4) focus on acquiring the key information needed for diagnosis and formulating an appropriate, prioritized differential diagnosis; individual competency #5 is taking advantage of second opinions, decision support, and checklists; and #6 is using reflection and critical thinking to improve diagnostic performance. Three competencies focus on teamwork: Involving the patient and family (#1) and all relevant health professionals (#2) in the diagnostic process; and (#3) ensuring safe transitions of care and handoffs, and "closing the loop" on test result communication. The final three competencies emphasize system-related aspects of care: (#1) Understanding how human-factor elements influence the diagnostic process; (#2) developing a supportive culture; and (#3) reporting and disclosing diagnostic errors that are recognized, and learning from both successful diagnosis and from diagnostic errors. Conclusions These newly defined competencies are relevant to all health professions education programs and should be incorporated into educational programs.

A Bottom Up Approach Towards Artificial Oxygenases by Combining Iron Coordination Complexes and Peptides.

Supramolecular systems resulting from the combination of peptides and a chiral iron coordination complex catalyze asymmetric epoxidation with aqueous hydrogen peroxide, providing good to excellent yields and high enantioselectivities in short reaction times. The peptide is shown to play a dual role; the terminal carboxylic acid assists the iron center in the efficient H2O2 activation step, while its ß-turn structure is crucial to induce high enantioselectivity in the oxygen delivering step. The high levels of stereoselection (84-92% ee) obtained by these supramolecular catalysts in the epoxidation of 1,1'-alkyl orthosubstituted styrenes, a notoriously challenging class of substrates for asymmetric catalysis, are not attainable with any other epoxidation methodology described so far. The current work combining an iron center ligated to N and O based ligands, and a peptide scaffold that shapes the second coordination sphere may be seen as a bottom up approach towards the design of artificial oxygenases.

From Substituent Effects to Applications: Enhancing the Optical Response of a Four-Component Assembly for Reporting EE Values.

High-throughput screening for asymmetric catalysts has stimulated an interest in optically-based enantiomeric-excess (ee) sensors, primarily for their improved time and cost efficiency when compared to the standard HPLC analysis. We present herein substituent-effect studies on a recently reported Zn(II) multicomponent assembly that is used for chiral, secondary alcohol ee detemination. The systematic altering of assemblies formed from select substituted pyridyl ligands pointed to the conclusion that steric effects dominate the mode of interaction at the pyridyl 3- and 6- positions. From these results we identified a new Zn(II)-centered multicomponent assembly with a higher dynamic range than previously reported. Calibration curves of the CD signals resulting from the new assembly led to an ee assay with a 1.7% error. To further the utility of the new assembly, a correlation was developed between alcohol substituent size to the respective enantiopure CD value.

Effect of Catheter Dwell Time on Risk of Central Line-Associated Bloodstream Infection in Infants.

BACKGROUND AND OBJECTIVE: Central venous catheters in the NICU are associated with significant morbidity and mortality because of the risk of central line-associated bloodstream infections (CLABSIs). The purpose of this study was to determine the effect of catheter dwell time on risk of CLABSI. METHODS: Retrospective cohort study of 13,327 infants with 15,567 catheters (93% peripherally inserted central catheters [PICCs], 7% tunneled catheters) and 256,088 catheter days cared for in 141 NICUs. CLABSI was defined using National Health Surveillance Network criteria. We defined dwell time as the number of days from line insertion until either line removal or day of CLABSI. We generated survival curves for each week of dwell time and estimated hazard ratios for CLABSI at each week by using a Cox proportional hazards frailty model. We controlled for postmenstrual age and year, included facility as a random effect, and generated separate models by line type. RESULTS: Median postmenstrual age was 29 weeks (interquartile range 26-33). The overall incidence of CLABSI was 0.93 per 1000 catheter days. Increased dwell time was not associated with increased risk of CLABSI for PICCs. For tunneled catheters, infection incidence was significantly higher in weeks 7 and 9 compared with week 1. CONCLUSIONS: Clinicians should not routinely replace uninfected PICCs for fear of infection but should consider removing tunneled catheters before week 7 if no longer needed. Additional studies are needed to determine what daily maintenance practices may be associated with decreased risk of infection, especially for tunneled catheters.

Function-Oriented Investigations of a Peptide-Based Catalyst that Mediates Enantioselective Allylic Alcohol Epoxidation.

We detail an investigation of a peptide-based catalyst 6 that is effective for the site- (>100:1:1) and enantioselective epoxidation (86% ee) of farnesol. Studies of the substrate scope exhibited by the catalyst are included, along with an exploration of optimized reaction conditions. Mechanistic studies are reported, including relative rate determinations for the catalyst and propionic acid, a historical perspective, truncation studies, and modeling using NMR data. Our compiled data advances our understanding of the inner workings of a catalyst that was identified through combinatorial means.