Impact of patient handover structure on neonatal perioperative safety.

OBJECTIVE: To compare the incidence, severity, preventability, and contributing factors of non-routine events-deviations from optimal care based on the clinical situation-associated with team-based, nurse-to-nurse, and mixed handovers in a large cohort of surgical neonates. STUDY DESIGN: A prospective observational study and one-time cross-sectional provider survey were conducted at one urban academic children's hospital. 130 non-cardiac surgical cases in 109 neonates who received pre- and post-operative NICU care. RESULTS: The incidence of clinician-reported NREs was high (101/130 cases, 78%) but did not differ significantly across acuity-tailored neonatal handover practices. National Surgical Quality Improvement-Pediatric occurrences of major morbidity were significantly higher (p < 0.001) in direct team handovers than indirect nursing or mixed handovers. CONCLUSIONS: NREs occur at a high rate and are of variable severity in neonatal perioperative care. NRE rates and contributory factors were homogenous across handover types. Surveyed clinicians recommend structured handovers for all patients at every transfer point regardless of acuity.

A polymorphism in the VKORC1 regulator calumenin predicts higher warfarin dose requirements in African Americans.

Warfarin demonstrates a wide interindividual variability in response that is mediated partly by variants in cytochrome P450 2C9 (CYP2C9) and vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1). It is not known whether variants in calumenin (CALU) (vitamin K reductase regulator) have an influence on warfarin dose requirements. We resequenced CALU regions in a discovery cohort of dose outliers: patients with high (>90th percentile, n = 55) or low (<10th percentile, n = 53) warfarin dose requirements (after accounting for known genetic and nongenetic variables). One CALU variant, rs339097, was associated with high doses (P = 0.01). We validated this variant as a predictor of higher warfarin doses in two replication cohorts: (i) 496 patients of mixed ethnicity and (ii) 194 African-American patients. The G allele of rs339097 (the allele frequency was 0.14 in African Americans and 0.002 in Caucasians) was associated with the requirement for a 14.5% (SD +/- 7%) higher therapeutic dose (P = 0.03) in the first replication cohort and a higher-than-predicted dose in the second replication cohort (allele frequency 0.14, one-sided P = 0.03). CALU rs339097 A>G is associated with higher warfarin dose requirements, independent of known genetic and nongenetic predictors of warfarin dose in African Americans.

Modification of cysteine.

This unit describes a number of methods for modifying cysteine residues of proteins and peptides by reduction and alkylation procedures. A general procedure for alkylation of cysteine residues in a protein of known size and composition with haloacyl reagents or N-ethylmaleimide (NEM) is presented, and alternate protocols describe similar procedures for use when the size and composition are not known and when only very small amounts of protein are available. Alkylations that introduce amino groups using bromopropylamine and N-(iodoethyl)-trifluoroacetamide are also presented. Two procedures that are often used for subsequent sequence analysis of the protein, alkylation with 4-vinylpyridine and acrylamide, are described, and a specialized procedure for 4-vinylpyridine alkylation of protein that has been adsorbed onto a sequencing membrane is also presented. Reversible modification of cysteine residues by way of sulfitolysis is described, and a protocol for oxidation with performic acid for amino acid compositional analysis is also provided. Gentle oxidation of cysteine residues to disulfides by exposure to air is detailed. Support protocols are included for recrystallization of iodoacetic acid, colorimetric detection of free sulfhydryls, and desalting of modified samples.

Edman sequencing as tool for characterization of synthetic peptides.

Sequence analysis of synthetic peptides using Edman chemistry can be very useful for the elucidation of certain types of synthetic problems, such as residue deletions and the presence of common stable derivatives, and for following the progress of the synthesis itself. However, it can also be a relatively poor technique for assessing quantitative aspects and the type and degree of adduct formation that arise from the synthetic chemistry. For these latter considerations, techniques such as mass spectrometry can often give more precise and informative data about the integrity of a synthetic peptide. Thus, sequence analysis is best applied judiciously and then used in combination with other methods. Furthermore, proper interpretation of the results of sequence analysis of synthetic peptides relies on a thorough knowledge of the sequencing process.

Affinity labeling in the presence of the reduced diphosphopyridine nucleotide NADH identifies peptides associated with the activities of human placental 3 beta-hydroxy-delta 5-steroid dehydrogenase/isomerase.

OBJECTIVE: We sought to identify peptides associated with activity in the primary structure of human placental 3 beta-hydroxy-delta 5-steroid dehydrogenase/isomerase (3 beta-HSD/isomerase). METHODS: Purified human placental 3 beta-HSD/isomerase was affinity-radioalkylated by 2 alpha-bromo [2'-14C]acetoxyprogesterone (2 alpha-[14C]BAP) in the presence or absence of the reduced diphosphopyridine nucleotide, NADH. NADH protected both 3 beta-HSD and isomerase from inactivation by 2 alpha-[14C]BAP. Tryptic peptides of unprotected and NADH-protected radioalkylated enzyme were purified by high-pressure liquid chromatography. The amino acid sequence of each radiolabeled peptide was determined and localized within the cDNA-derived primary structure of the enzyme. RESULTS: According to the sequence analyses, NADH shifted radioalkylation by 2 alpha-[14C]BAP away from the Arg-250 peptide (251GQFYYISDDTPHQSYDNLNYTLSK274) and toward the Lys-135 tryptic peptide (136EIIQNGHEEEPLENTWPAPYPHSK159). Based on amino acid analysis to quantitate radioactivity incorporated per nmol peptide, NADH decreased the radiolabeling of His262 in the Arg-250 peptide by 8.2-fold. His142 in the Lys-135 peptide was radiolabeled by 2 alpha-[14C]BAP only in the presence of NADH. CONCLUSIONS: We have previously reported that the substrate pregnenolone blocks the inactivation of 3 beta-HSD by 2 alpha-[14C]BAP through the protection of His262 in the Arg-250 peptide. Protection by NADH against the inactivation of isomerase as well as 3 beta-HSD is evidence that 2 alpha-[14C]BAP binds at the active sites of both enzyme activities. Because the same Arg-250 peptide has been affinity-alkylated in studies that targeted each of the two activities, we propose that the 3 beta-HSD and isomerase reactions are catalyzed in this region of the enzyme protein.

Affinity radiolabeling identifies peptides and amino acids associated with substrate binding in human placental 3 beta-hydroxy-delta(5)-steroid dehydrogenase.

Purified human placental 3 beta-hydroxy-delta(5)-steroid dehydrogenase (3 beta-HSD) was affinity radiolabeled by 2 alpha-bromo[2'-14C]acetoxyprogesterone (2 alpha-BAP) in the presence or absence of 3 beta-HSD substrate, pregnenolone. The substrate steroid substantially protects 3 beta-HSD activity from inactivation by 2 alpha-BAP. Tryptic peptides of unprotected and substrate-protected radioalkylated enzyme were purified by high pressure liquid chromatography. The amino acid sequence of each radiolabeled peptide was determined and localized within the cDNA-derived primary structure of the enzyme. According to the percent total radioactivity associated with each of four radiolabeled peaks separated by high pressure liquid chromatography, two peptides were protected by substrate from affinity radioalkylation by 2 alpha-BAP. The first, 251GQFYYISDDTPHQSYDNLNYTLSK274, was produced by tryptic cleavage at Arg-250 and Lys-274 (the Arg-250 peptide) and contained radiolabeled His262. The second, 176NGGTLYTCALR186, was produced by tryptic cleavage at Lys-175 and Arg-186 (the Lys-175 peptide) and contained radiolabeled Cys183. Based on amino acid analysis to quantitate radioactivity incorporated per nmol of peptide, substrate steroid decreased the radiolabeling of His262 in the Arg-250 peptide by 3.6-fold and decreased the radiolabeling of Cys183 in the Lys-175 peptide by 3.7-fold. Three minor radiolabeled peptides (the NH2-terminal, Arg-71, and Arg-196 tryptic peptides) were also identified in the primary structure, but pregnenolone did not diminish their affinity radioalkylation. These observations indicate that the Arg-250 and Lys-175 peptides are involved in substrate binding and suggest that His262 and Cys183 are in close proximity in the three-dimensional structure of the enzyme.