Comparing risk-adjustment methods for provider profiling

Risk-adjustment and provider profiling have become common terms as the medical profession attempts to measure quality and assess value in health care. One of the areas of care most thoroughly developed in this regard is quality assessment for coronary artery bypass grafting (CABG). Because in-hospital mortality following CABG has been studied extensively, risk-adjustment mechanisms are already being used in this area for provider profiling. This study compares eight different risk-adjustment methods as applied to a CABG surgery population of 28 providers. Five of the methods use an external risk-adjustment algorithm developed in an independent population, while the other three rely on an internally developed logistic model. The purposes of this study are to: (i) create a common metric by which to display the results of these various risk-adjustment methodologies with regard to dichotomous outcomes such as in-hospital mortality, and (ii) to compare how these risk-adjustment methods quantify the 'outlier' standing of providers. Section 2 describes the data, the external and internal risk-adjustment algorithms, and eight approaches to provider profiling. Section 3 then demonstrates the results of applying these methods on a data set specifically collected for quality improvement.     

The effect of point mutations on the free energy of transmembrane alpha-helix dimerization

Glycophorin A forms homodimers through interaction of the single, helical transmembrane domains of the monomers. The dimers are stable in sodium dodecylsulfate (SDS), permitting a number of studies that have identified a critical motif of residues that mediates dimer formation. We have used analytical ultracentrifugation to measure the energy of dimerization in a non-denaturing detergent solution and have observed the changes in energy arising from two of the mutants previously studied. Use of the detergent pentaoxyethylene octyl ether (C8E5) is a great advantage, since its micelles are neutrally buoyant and the detergent allows a reversible association to occur between monomer and dimer states of the glycophorin A transmembrane helices during the time-scale of sedimentation equilibrium. Use of this detergent in analytical ultracentrifugation may enable a wide range of studies of molecular association events in membrane proteins. We find that the glycophorin A transmembrane helix dimerizes with a dissociation constant of 240(+/-50) nM, corresponding to a free energy of dissociation of 9.0(+/-0.1) kcal mol-1. Point mutants that were found to be disruptive in SDS (L75A, I76A) reduced the dimer affinity in the C8E5 detergent environment (Kd=1.7(+/-0.2) microM and 4.2(+/-0.9) microM, respectively). Thus, the earlier findings are placed on a quantitative, relative energy scale of association by our measurements. Molecular modeling and simulations suggest that the energy differences can be accounted for as changes in van der Waals interactions between helices.     

A stereotaxic template atlas of the macaque brain for digital imaging and quantitative neuroanatomy

A stereotaxic brain atlas of the longtailed macaque (Macaca fascicularis) is presented in a format suitable for use as a template atlas of the macaque brain. It includes most of the brain segmented to show the boundaries of landmark structures such that every point in the brain can be represented by a unique set of coordinates in three-dimensional space and ascribed unambiguously to one and only one primary structure. More than 400 structures are represented, including 360 volumetric structures, which constitute the substance of the brain, and 50 superficial features. To facilitate use with ventriculography, magnetic resonance imaging, and other noninvasive imaging techniques, the stereotaxic space is referenced to internal landmarks, viz., the anterior commissure and posterior commissure; the center of the anterior commissure at the midline is the origin of the stereotaxic axes. Reference of stereotaxis to this bicommissural space facilitates structural comparison with human brain atlases, which are commonly referenced to the biocommissural line. It also facilitates comparison of brains of different nonhuman primate species by providing a template brain against which to compare size and internal variability. Thirty-three coronal sections at 1-mm intervals from the spinomedullary junction to the rostral extreme of the caudate nucleus show most structures of the hindbrain, midbrain, and subcortical forebrain. Separately, four side views and 16 coronal sections show cortical structures. Structures are represented by outlines of their boundaries and labeled according to NeuroNames, a systematic English nomenclature of human and nonhuman primate neuroanatomy. Abbreviations are based on a protocol designed to facilitate cross-species comparisons. Instructions are provided for: (1) locating sites from the Template Atlas in the conventional stereotaxic space of an experimental animal, (2) locating sites identified by conventional stereotaxis in the Template Atlas, and (3) using the Template Atlas to collate, compare, and display image information (e.g., labeled cells, recording sites, stimulation sites, lesions) from multiple animals.     

Investigation of the effects of antisense oligodeoxynucleotides to islet amyloid polypeptide mRNA on insulin release, content and expression

We have investigated the effects of antisense oligodeoxynucleotides (oligos) to islet amyloid polypeptide (IAPP) mRNA on the expression and secretion of IAPP and insulin, in the clonal beta-cell line HIT-T15. Phosphorothioate-modified oligos were cytotoxic compared with phosphodiester (D)-oligos. Of the nine oligos tested using a lipofection reagent, O3, a 30-mer D-oligo complementary to a sequence downstream of the IAPP initiation codon, showed a significant dose-dependent suppression of IAPP mRNA, with a 42% decrease at 7.5 microM, compared with a scrambled (MSO3) control oligo (n = 3, P < 0.01). A subsequent 89% suppression of IAPP release was observed in the 4-h period following antisense treatment (1.78 +/- 0.13 (MSO3) vs 0.19 +/- 0.14 (O3) pmol/10(6) cells per 240 min, n = 7, P < 0.01). A significant increase in insulin mRNA (100 +/- 10% (MSO3) vs 124 +/- 8% (O3), n = 3, P < 0.05) and insulin content (13.0 +/- 0.9 (MSO3) vs 17.4 +/- 1.4 (O3) pmol/10(6) cells, n = 7, P = 0.028) was observed following treatment with O3 at 7.5 microM. O8, a 20-mer D-oligo directed to a region of IAPP mRNA further downstream than O3, also showed a decrease in IAPP mRNA and peptide release and an increase in insulin content. No significant changes were observed in the expression and release of the unrelated beta-cell peptide, neuropeptide Y. We thus show a suppression of synthesis and release of IAPP in HIT-T15 cells using antisense oligos. The associated increase in insulin mRNA and content in these cells after treatment with IAPP antisense oligos is in accord with an inhibitor action of IAPP on insulin availability.     

The structure-selectivity and sequence-preference of the junction-resolving enzyme CCE1 of Saccharomyces cerevisiae

CCE1 is a DNA junction-resolving enzyme involved in the resolution of recombining mitochondrial DNA in Saccharomyces cerevisiae. The CCE1 gene was cloned by PCR, and the expressed protein purified to homogeneity. CCE1 was found to bind to four-way DNA junctions, with a strong structural selectivity. The enzyme binds DNA junctions as a dimer, with slow subunit exchange occurring in free solution. While CCE1 binds equally to synthetic four-way DNA junctions of any sequence, it exhibits pronounced sequence-selectivity in cleavage. Both fixed junctions and those capable of branch migration can be cleaved, with a preference for cleavage at the sequence 5'CT/. Cleavage of junctions tethered to adopt specific stacking isomers demonstrated that the target sequences are cleaved fivefold faster when located on a continuous strand compared to an exchanging strand.     

Expression and regulation of constitutive and acute phase serum amyloid A mRNAs in hepatic and non-hepatic cell lines

A 60 kb conjugative plasmid, pND300, which encodes nisin resistance, was identified in Lactococcus lactis ssp. lactis (L. lactis) M189. pND300 was found to mobilize the transfer of some other plasmids as indicated by the mobilization of plasmids encoding lactose utilization. The nisin resistance determinant from pND300 was initially subcloned on a 12 kb DNA fragment and subsequently reduced to 10.4 kb. Restriction analysis, PCR, Southern hybridization and sequencing illustrated that the nisin resistance of pND300 is very similar to that encoded by the transposon involved in nisin production. pND300 encodes nisR as well as nisK and the recently reported nisF, nisE and nisG, but does not encode nisI. The DNA fragment encoding the nis genes is flanked by IS946 with a copy at each end in reverse orientation. The expression of these nis genes is probably controlled by a putative promoter upstream of nisR, which is composed of the TTGCAA hexanucleotide on the insertion sequence IS946 and the TATAAT sequence 21 bp downstream.     

Pathways of glutathione metabolism and transport in isolated proximal tubular cells from rat kidney

Cellular uptake and metabolism of exogenous glutathione (GSH) in freshly isolated proximal tubular (PT) cells from rat kidney were examined in the absence and presence of inhibitors of GSH turnover [acivicin, L-buthionine-S,R-sulfoximine (BSO)] to quantify and assess the role of different pathways in the handling of GSH in this renal cell population. Incubation of PT cells with 2 or 5 mM GSH in the presence of acivicin/BSO produced 3- to 4-fold increases in intracellular GSH within 10-15 min. These significantly higher intracellular concentrations were maintained for up to 60 min. At lower concentrations of extracellular GSH, an initial increase in intracellular GSH concentrations was observed, but this was not maintained for the 60-min time course. In the absence of inhibitors, intracellular concentrations of GSH increased to levels that were 2- to 3-fold higher than initial values in the first 10-15 min, but these dropped below initial levels thereafter. In both the absence and presence of acivicin/BSO, PT cells catalyzed oxidation of GSH to glutathione disulfide (GSSG) and degradation of GSH to glutamate and cyst(e)ine. Exogenous tert-butyl hydroperoxide oxidized intracellular GSH to GSSG in a concentration-dependent manner and extracellular GSSG was transported into PT cells, but limited intracellular reduction of GSSG to GSH occurred. Furthermore, incubation of cells with precursor amino acids produced little intracellular synthesis of GSH, suggesting that PT cells have limited biosynthetic capacity for GSH under these conditions. Hence, direct uptake of GSH, rather than reduction of GSSG or resynthesis from precursors, may be the primary mechanism to maintain intracellular thiol redox status under toxicological conditions. Since PT cells are a primary target for toxicants, the ability of these cells to rapidly take up and metabolize GSH may serve as a defensive mechanism to protect against chemical injury.