Peeling back the layers: First phylogenomic insights into the Ledebouriinae (Scilloideae, Asparagaceae).

The Ledebouriinae (Scilloideae, Asparagaceae) are a widespread group of bulbous geophytes found predominantly throughout seasonal climates in sub-Saharan Africa, with a handful of taxa in Madagascar, the Middle East, India, and Sri Lanka. Phylogenetic relationships within the group have been historically difficult to elucidate. Here, we provide the first phylogenomic perspective into the Ledebouriinae. Using the Angiosperms353 targeted enrichment probe set, we consistently recovered four major clades (i.e., two Ledebouria clades, Drimiopsis, and Resnova). The two Ledebouria clades closely align with geography, either consisting almost entirely of sub-Saharan African taxa (Ledebouria Clade A), or East African and non-African taxa (Ledebouria Clade B). Our results suggest that the Ledebouriinae likely underwent a rapid radiation leading to rampant incomplete lineage sorting. We additionally find evidence for potential historical hybridization between Drimiopsis and a subclade within Ledebouria Clade A.

Denaturant-dependent conformational changes in a beta-trefoil protein: global and residue-specific aspects of an equilibrium denaturation process.

Conformational properties of the folded and unfolded ensembles of human interleukin-1 receptor antagonist (IL-1ra) are strongly denaturant-dependent as evidenced by high-resolution two-dimensional nuclear magnetic resonance (NMR), limited proteolysis, and small-angle X-ray scattering (SAXS). The folded ensemble was characterized in detail in the presence of different urea concentrations by (1)H-(15)N HSQC NMR. The beta-trefoil fold characteristic of native IL-1ra was preserved until the unfolding transition region beginning at 4 M urea. At the same time, a subset of native resonances disappeared gradually starting at low denaturant concentrations, indicating noncooperative changes in the folded state. Additional evidence of structural perturbations came from the chemical shift analysis, nonuniform and bell-shaped peak intensity profiles, and limited proteolysis. In particular, the following nearby regions of the tertiary structure became progressively destabilized with increasing urea concentrations: the beta-hairpin interface of trefoils 1 and 2 and the H2a-H2 helical region. These regions underwent small-scale perturbations within the native baseline region in the absence of populated molten globule-like states. Similar regions were affected by elevated temperatures known to induce irreversible aggregation of IL-1ra. Further evidence of structural transitions invoking near-native conformations came from an optical spectroscopy analysis of its single-tryptophan variant W17A. The increase in the radius of gyration was associated with a single equilibrium unfolding transition in the case of two different denaturants, urea and guanidine hydrochloride (GuHCl). However, the compactness of urea- and GuHCl-unfolded molecules was comparable only at high denaturant concentrations and deviated under less denaturing conditions. Our results identified the role of conformational flexibility in IL-1ra aggregation and shed light on the nature of structural transitions within the folded ensembles of other beta-trefoil proteins, such as IL-1beta and hFGF-1.

Biophysical characterization of structural properties and folding of interleukin-1 receptor antagonist.

Structural properties and folding of interleukin-1 receptor antagonist (IL-1ra), a therapeutically important cytokine with a symmetric beta-trefoil topology, are characterized using optical spectroscopy, high-resolution NMR, and size-exclusion chromatography. Spectral contributions of two tryptophan residues, Trp17 and Trp120, present in the wild-type protein, have been determined from mutational analysis. Trp17 dominates the emission spectrum of IL-1ra, while Trp120 is quenched presumably by the nearby cysteine residues in both folded and unfolded states. The same Trp17 gives rise to two characteristic negative peaks in the aromatic CD. Urea denaturation of the wild-type protein is probed by measuring intrinsic and extrinsic (binding of 1-anilinonaphthalene-8-sulfonic acid) fluorescence, near- and far-UV CD, and 1D and 2D ((1)H-(15)N heteronuclear single quantum coherence (HSQC)) NMR. Overall, the data suggest an essentially two-state equilibrium denaturation mechanism with small, but detectable structural changes within the pretransition region. The majority of the (1)H-(15)N HSQC cross-peaks of the folded state show only a limited chemical shift change as a function of the denaturant concentration. However, the amide cross-peak of Leu31 demonstrates a significant urea dependence that can be fitted to a two-state binding model with a dissociation constant of 0.95+/-0.04 M. This interaction has at least a five times higher affinity than reported values for nonspecific urea binding to denatured proteins and peptides, suggesting that the structural context around Leu31 stabilizes the protein-urea interaction. A possible role of denaturant binding in inducing the pretransition changes in IL-1ra is discussed. Urea unfolding of wild-type IL-1ra is sufficiently slow to enable HPLC separation of folded and unfolded states. Quantitative size-exclusion chromatography has provided a hydrodynamic view of the kinetic denaturation process. Thermodynamic stability and unfolding kinetics of IL-1ra resemble those of structurally and evolutionary close IL-1beta, suggesting similarity of their free energy landscapes.

Discovery of ligands for Nurr1 by combined use of NMR screening with different isotopic and spin-labeling strategies.

A comprehensive approach to target screening, hit validation, and binding site determination by nuclear magnetic resonance (NMR) spectroscopy is presented. NMR (19)F signal perturbation was used to screen a small compound library and identify candidate ligands to the target of interest. Ligand dissociation constants were measured using a pegylated form of the protein, which resulted in a 2-fold increase in the strength of the saturation transfer difference signal. The initial small-molecule hits were further optimized by combining a residue-specific labeling strategy, to identify the specific sites of interaction with the protein, with a second site screening approach based on relaxation enhancement using a paramagnetic probe. The advantages of this combination strategy in the identification and optimization of weak binding chemical entities early in a program are illustrated with the discovery of a low micromolar ligand (K(d) = 20 microM) for Nurr1 and identification of the binding site location through residue-specific (15)N isotope labeling and derivatization of Cys residues with 2-mercaptoethanol-1-(13)C.

Addison's disease and the regulation of potassium: the role of insulin and aldosterone.

It is proposed that insulin has a cardinal role in the regulation of serum potassium levels in man, which may be of greater importance than the effect of insulin on glucose metabolism. Although the first described action of insulin was on glucose transport, it is a hormone with many functions some of which may operate in a metabolic hierarchy depending on the relative importance of the action required. Insulin also promotes the transport of potassium ions from the extracellular space to the intracellular space and it is suggested that there are occasions where this action may take place at the expense of glucose regulation. In metabolic terms, tight control of serum potassium is of greater importance than precise control of serum glucose, because quite small variations in serum potassium may cause death whereas wide variations in serum glucose may be tolerated. Serum potassium levels generally remain very stable despite large daily variations in potassium intake. It follows that potassium control mechanisms must be of outstanding efficiency as serious disturbances of potassium balance are relatively uncommon. 'Nature makes experiments on Man': shadowy but important physiological mechanisms that may almost be taken for granted in normal health are often brightly illuminated by unusual pathological conditions. This paper describes two remarkable patients who presented with extreme hyperkalaemia. This condition was the result of simultaneous insulin and aldosterone deficiency occurring because of concomitant diabetes and Addison's disease. Other medical conditions with disturbances in aldosterone, insulin and potassium control will be referred to in support of the hypothesis that insulin secretion is central to potassium regulation. This hypothesis explains the secondary disturbances in glucose metabolism that occurs in clinical situations where the primary problem is perturbation of potassium regulation.

Application of statistical process control to physician-specific emergency department patient satisfaction scores: a novel use of the funnel plot.

OBJECTIVES: Emergency department (ED) patient satisfaction remains a high priority for many hospitals. Patient surveys are a common tool for measuring patient satisfaction, and process improvement efforts are aimed at improving patient satisfaction scores. In some institutions, patient satisfaction scores can be calculated for each emergency physician (EP). ED leaders are faced with the task of interpreting individual as well as group physician scores to identify opportunities for improvement. Analysis of these data can be challenging because of the relatively small numbers of returned surveys assignable to a single physician, variable numbers of surveys returned for each physician and high standard deviations (SDs) for individual physician scores. The objective was to apply statistical process control methodology to analyze individual as well as group physician patient satisfaction scores. The novel use of funnel plots to interpret individual physician patient satisfaction scores, track individual physician scores over two successive 8-month periods, and monitor physician group performance is demonstrated. METHODS: Patient satisfaction with physicians was measured using Press Ganey surveys for a 65,000-volume ED over two successive 8-month periods. Using funnel plots, individual physician patient satisfaction scores were plotted against the number of surveys completed for each physician for each 8-month period. Ninety-fifth and 99th percentile control limits were displayed on the funnel plots to illustrate individual physician patient satisfaction scores that are within, versus those that are outside of, expected random variation. Control limits were calculated using mean patient satisfaction scores and SDs for the entire group of physicians. Additional funnel plots were constructed to demonstrate changes in individual physicians' patient satisfaction scores as a function of increasing numbers of returned surveys and to illustrate changes in the group's patient satisfaction scores between the first and second 8-month intervals after the institution of process improvement efforts aimed at improving patient satisfaction. RESULTS: For the first 8-month period, 34,632 patients were evaluated in and discharged from the ED, with 581 surveys returned for 21 physicians. The mean (±SD) overall group physician patient satisfaction score was 81.8 (±24.7). Returned surveys per physician ranged from 2 to 58. For the second period, 34,858 patients were evaluated and discharged from the ED, with 670 patient satisfaction surveys returned for 20 physicians. The mean (±SD) overall physician score for all surveys returned during the second period was 85.0 (±22.2). Returned surveys per physician ranged from 8 to 65. CONCLUSIONS: The application of statistical control methodology using funnel plots as a means of analyzing ED group and physician patient satisfaction scores was possible. The authors believe that using funnel plots to analyze scores graphically can rapidly help determine the significance of individual physician patient satisfaction scores. In addition, serial funnel plots may prove to be useful as a means of measuring changes in patient satisfaction, particularly in response to quality improvement interventions.

Raman tweezers and their application to the study of singly trapped eukaryotic cells.

In this review the recent emergence of Raman tweezers as an analytical technique for single eukaryotic cell analysis is described. The Raman tweezer technique combines Raman spectroscopy as a diagnostic tool with optical tweezers by which means single cells can be trapped and manipulated in a laser beam using a high numerical aperture imaging microscope. Necessary instrumental requirements to facilitate Raman tweezer experiments are discussed together with practical considerations such as the potential for photodamage of cells subjected to trapping and Raman excitation. Specific applications of Raman tweezers to the analysis of cancer cells, erythrocytes and lymphocytes, micro-organisms and sub-cellular components e.g.chromosomes and mitochondria are then discussed followed by a summary of the future potential of the technique for single cell analysis.

Hepcidin revisited, disulfide connectivity, dynamics, and structure.

Hepcidin is a tightly folded 25-residue peptide hormone containing four disulfide bonds, which has been shown to act as the principal regulator of iron homeostasis in vertebrates. We used multiple techniques to demonstrate a disulfide bonding pattern for hepcidin different from that previously published. All techniques confirmed the following disulfide bond connectivity: Cys(1)-Cys(8), Cys(3)-Cys(6), Cys(2)-Cys(4), and Cys(5)-Cys(7). NMR studies reveal a new model for hepcidin that, at ambient temperatures, interconverts between two different conformations, which could be individually resolved by temperature variation. Using these methods, the solution structure of hepcidin was determined at 325 and 253 K in supercooled water. X-ray analysis of a co-crystal with Fab appeared to stabilize a hepcidin conformation similar to the high temperature NMR structure.

Structural and thermodynamic effects of ANS binding to human interleukin-1 receptor antagonist.

Although 8-anilinonaphthalene-1-sulfonic acid (ANS) is frequently used in protein folding studies, the structural and thermodynamic effects of its binding to proteins are not well understood. Using high-resolution two-dimensional NMR and human interleukin-1 receptor antagonist (IL-1ra) as a model protein, we obtained detailed information on ANS-protein interactions in the absence and presence of urea. The effects of ambient to elevated temperatures on the affinity and specificity of ANS binding were assessed from experiments performed at 25 degrees C and 37 degrees C. Overall, the affinity of ANS was lower at 37 degrees C compared to 25 degrees C, but no significant change in the site specificity of binding was observed from the chemical shift perturbation data. The same site-specific binding was evident in the presence of 5.2 M urea, well within the unfolding transition region, and resulted in selective stabilization of the folded state. Based on the two-state denaturation mechanism, ANS-dependent changes in the protein stability were estimated from relative intensities of two amide resonances specific to the folded and unfolded states of IL-1ra. No evidence was found for any ANS-induced partially denatured or aggregated forms of IL-1ra throughout the experimental conditions, consistent with a cooperative and reversible denaturation process. The NMR results support earlier observations on the tendency of ANS to interact with solvent-exposed positively charged sites on proteins. Under denaturing conditions, ANS binding appears to be selective to structured states rather than unfolded conformations. Interestingly, the binding occurs within a previously identified aggregation-critical region in IL-1ra, thus providing an insight into ligand-dependent protein aggregation.

Discovery of N-phenyl nicotinamides as potent inhibitors of Kdr.

Inhibition of tumor-induced angiogenesis is a promising strategy in anticancer research. Neovascularization is a process required for both tumor growth and metastasis. Enhanced understanding of the underlying molecular mechanisms has led to the discovery of a variety of pharmaceutically attractive targets. Decades of investigation suggest that vascular endothelial growth factor (VEGF) and its receptors, in particular VEGFR2 or kinase insert-domain-containing receptor (Kdr), play a critical role in the growth and survival of endothelial cells in newly forming vasculature. The clinical utility of inhibitors of this receptor tyrosine kinase is currently under intense investigation. Herein we report our efforts in this arena.

The sharpened Romberg test for assessing ataxia in mild acute mountain sickness.

OBJECTIVE: To evaluate the Sharpened Romberg Test (SRT) as a measure of ataxia in subjects with mild acute mountain sickness in order to determine its sensitivity and specificity. METHODS: The SRT was performed in 23 subjects during ascent to 5260 m. RESULTS: The SRT was more often abnormal than the traditional heel-to-toe test, and at the highest altitude it was related to higher median Lake Louise symptom scores with predictive values of 60% sensitivity and 89% specificity. Our evaluation of the SRT appears to agree with similar studies on ataxia showing a lack of correlation between ataxia and symptoms of acute mountain sickness at altitudes below 5260 m. CONCLUSION: The SRT was easy to perform and provided a quantitative assessment of truncal ataxia in the field without the need for specialized equipment.

Design and synthesis of potent pyridazine inhibitors of p38 MAP kinase.

Novel potent trisubstituted pyridazine inhibitors of p38 MAP (mitogen activated protein) kinase are described that have activity in both cell-based assays of cytokine release and animal models of rheumatoid arthritis. They demonstrated potent inhibition of LPS-induced TNF-alpha production in mice and exhibited good efficacy in the rat collagen induced arthritis model.

Characterization of components of the mismatch repair machinery in Trypanosoma brucei.

Mismatch repair is one of a number of DNA repair pathways that cells possess to deal with damage to their genome. Mismatch repair is concerned with the recognition and correction of incorrectly paired bases, which can be base-base mismatches or insertions or deletions of a few bases, and appears to have been conserved throughout evolution. Primarily, this is concerned with increasing the fidelity of DNA replication, but also has important roles in the regulation of homologous recombination and the correction of chemical damage. In this study, we describe five genes in the protistan parasite Trypanosoma brucei that are likely to be involved in nuclear mismatch repair. The predicted T. brucei mismatch repair genes are diverged compared with their likely counterparts in the other eukaryotes examined to date. To demonstrate that these do indeed encode a functional nuclear mismatch repair system, we made T. brucei null mutants in two of the genes, MSH2 and MLH1, that are likely to be central to the functioning of the mismatch repair machinery. These mutations resulted in increased rates of sequence variation at a number of microsatellite loci in the parasite genome, and led to increased tolerance to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine, both phenotypes consistent with mismatch repair impairment.

Serum test for assessment of patients with Bence Jones myeloma.

Bence Jones protein in urine (immunoglobulin free-light-chains) is characteristic of light-chain multiple myeloma. We aimed to compare a quantitative immunoassay for serum free-light-chains with urine tests. Of 224 patients with light-chain myeloma tested at entry to clinical trials, all were correctly identified from serum samples. During monitoring of 82 patients, changes in serum and urine free-light-chains corresponded, but urine became negative for free-light-chains in 26 patients, whereas it remained abnormal in serum in 73 patients. Serum assays could replace Bence Jones protein urine tests for patients with light-chain multiple myeloma.