Epitope mapping using mRNA display and a unidirectional nested deletion library

In vitro selection targeting an anti-polyhistidine monoclonal antibody was performed using mRNA display with a random, unconstrained 27-mer peptide library. After six rounds of selection, epitope-like peptides were identified that contain two to five consecutive, internal histidines and are biased for arginine residues, without any other identifiable consensus. The epitope was further refined by constructing a high-complexity, unidirectional fragment library from the final selection pool. Selection by mRNA display minimized the dominant peptide from the original selection to a 15-residue functional sequence (peptide Cmin: RHDAGDHHHHHGVRQ; K-D = 38 nM). Other peptides recovered from the fragment library selection revealed a separate consensus motif (ARRXA) C-terminal to the histidine track. Kinetics measurements made by surface plasmon resonance, using purified Fab (antigen-binding fragment) to prevent avidity effects, demonstrate that the selected peptides bind with 10- to 75-fold higher affinities than a hexahistidine peptide. The highest affinity peptides (K-D approximate to 10 nM) encode both a short histidine track and the ARRXA motif, suggesting that the motif and other flanking residues make important contacts adjacent to the core polyhistidine-binding site and can contribute > 2.5 kcal/mol of binding free energy. The fragment library construction methodology described here is applicable to the development of high-complexity protein or cDNA expression libraries for the identification of protein-protein interaction domains

A Peptide Core Motif for Binding to Heterotrimeric G Protein α Subunits

Recently, in vitro selection using mRNA display was used to identify a novel peptide sequence that binds with high affinity to G{alpha}i1. The peptide was minimized to a 9-residue sequence (R6A-1) that retains high affinity and specificity for the GDP-bound state of G{alpha}i1 and acts as a guanine nucleotide dissociation inhibitor (GDI). Here we demonstrate that the R6A-1 peptide interacts with G{alpha} subunits representing all four G protein classes, acting as a core motif for G{alpha} interaction. This contrasts with the consensus G protein regulatory(GPR) sequence, a 28-mer peptide GDI derived from the GoLoco (G{alpha}i/0-Loco interaction)/GPR motif that shares no homology with R6A-1 and binds only to G{alpha}i1-3 in this assay. Binding of R6A-1 is generally specific to the GDP-bound state of the G{alpha} subunits and excludes association with G{beta}{gamma}. R6A-G{alpha}i1 complexes are resistant to trypsin digestion and exhibit distinct stability in the presence of Mg2+, suggesting that the R6A and GPR peptides exert their activities using different mechanisms. Studies using G{alpha}i1/G{alpha}s chimeras identify two regions of G{alpha}i1 (residues 1–35 and 57–88) as determinants for strong R6A-Gi{alpha}1 interaction. Residues flanking the R6A-1 peptide confer unique binding properties, indicating that the core motif could be used as a starting point for the development of peptides exhibiting novel activities and/or specificity for particular G protein subclasses or nucleotide-bound states

A Study by Nitrogen-15 Nuclear Magnetic Resonance Spectroscopy of the State of Histidine in the Catalytic Triad of α-Lytic Protease

Hydrolytic cleavage of peptide bonds is an energetically· favorable reaction, but one that normally takes place very slowly at room temperature, even in the presence of rather strong acids or bases. It can be strongly catalyzed by many proteases, and much effort has been expended to determine how these have the ability to increase the rate of hydrolysis by a million-fold or more in neutral solutions. One of the types of proteases, the serine-protease family, is characterized by the presence at the active site of a "catalytic triad" comprised of the side-chain residues of serine, histidine and aspartic acid

Left main coronary artery originating from the right sinus of valsalva and coursing between the aorta and pulmonary trunk

Findings are described in five patients who at necropsy were found to have origin of the left main coronary artery from the right sinus of Valsalva and coursing of the anomalously arising artery between aorta and pulmonary trunk to reach the left side of the heart. Three of the five patients were boys and died suddenly at ages 13, 14 and 19 years, respectively: two of them had had one or more episodes of syncope and the third had an abnormal electrocardiogram. The fourth patient, a 64 year old woman, died of chronic congestive heart failure 1 year after an acute myocardial infarction. She had insignificant coronary atherosclerosis. The fifth patient, an 81 year old man, died of chronic alcoholism, having been free of symptoms of cardiac dysfunction during life.Additionally, clinical and necropsy findings are summarized in 38 previously reported necropsy patients with the coronary anomaly. Of these 38 (34 male [89%]), 23 (61 %) died suddenly in the first two decades of life; death in 6 others (16%) appears to have been related to coronary atherosclerosis and 9 patients (24%) died from noncoronary causes. Thus, this anomaly is life-threatening. Why it frequently causes fatal cardiac arrest in some young individuals and allows a normal life span in others remains unclear

Dynamical age of solar wind turbulence in the outer heliosphere

In an evolving turbulent medium, a natural timescale can be defined in terms of the energy decay time. The time evolution may be complicated by other effects such as energy supply due to driving, and spatial inhomogeneity. In the solar wind the turbulence appears not to be simply engaging in free decay, but rather the energy level observed at a particular position in the heliosphere is affected by expansion, “mixing,” and driving by stream shear. Here we discuss a new approach for estimating the “age” of solar wind turbulence as a function of heliocentric distance, using the local turbulent decay rate as the natural clock, but taking into account expansion and driving effects. The simplified formalism presented here is appropriate to low cross helicity (non-Alfvénic) turbulence in the outer heliosphere especially at low helio-latitudes. We employ Voyager data to illustrate our method, which improves upon the familiar estimates in terms of local eddy turnover times

Importance of Perioperative Processes of Care for Length of Hospital Stay after Laparoscopic Surgery

Background and Purpose: The technologic imperative has prompted the adoption of complex laparoscopic techniques by physicians with various degrees of skill. We sought to measure the impact of both case mix and physician practice (perioperative process/risk factors) on length of stay (LOS)—a common benchmark— after laparoscopic surgery. Patients and Methods: We identified 911 patients undergoing laparoscopic retroperitoneal surgery between 1996 and 2004, who comprise our study population. Patients remaining in the hospital >5 days—the 90th percentile for the sample—were classified as having a prolonged LOS. Adjusted models were developed to determine the independent association of case mix and process measures with a prolonged LOS. The likelihood ratio test was used to discern the improvement of fit of the process model compared with the case-mix model. Results: Among factors related to case mix and structure of care, increasing age (odds ratio [OR] 1.1; 95% CI 1.0, 1.2), less surgeon experience (OR 6.1; 95% CI 2.1, 17.2), male gender (OR 2.1; 95% CI 1.2, 4.0), and American Society of Anesthesiologists score of 3 or 4 (OR 7.2; 95% CI 2.2, 23.3) were independently associated with a prolonged LOS. The need for a transfusion (OR 9.4; 95% CI 33.9, 23.2), the development of a postoperative complication (OR 4.6; 95% CI 2.2, 9.5), and longer operative time (OR 1.5; 95% CI 1.3, 1.8) explained additional variation in prolonged LOS outcomes when considering perioperative process/risk factors in the model. Perioperative factors significantly improved the fit of the model (χ 2 statistic 101.8; p < 0.0001). Conclusions: Significant variation in outcomes is explained by factors describing aspects of surgical expertise. Variability in the surgical skill set is likely greatest during the laparoscopic learning curve, which raises a quality-of-care concern during the initial implementation of the technique. Policies attempting to smooth the laparoscopic learning curve, such as mentoring and skill measurement prior to credentialing, could improve the quality of care.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63271/1/end.2006.20.776.pd

Failure After Laparoscopic Pyeloplasty: Prevention and Management

Background and Purpose: Because of the high success of laparoscopic pyeloplasty (LP) for ureteropelvic junction obstruction, strategies for managing failures are less well described. We report our experience with persistent or recurrent obstruction after LP. Patients and Methods: We reviewed 128 patients who were treated with LP at our institution from 1996 through 2008. Success was defined as objective resolution of obstruction by renal scintigraphy, Whitaker testing, or direct visualization. We extracted data by chart review regarding patient demographics, medical history, operative technique, and salvage treatments. We then assessed for association between patient characteristics and treatment failure. Results: Overall, 102 patients had sufficient follow-up, of which 84 (82%) were successes. Of 18 failures, median time to failure was 2.5 months (0.5-88-mos). Of 10 failures managed endoscopically, 7 were salvaged. One of two patients treated conservatively ultimately had resolution while six patients needed simple nephrectomy. Overall, 8 (44%) were salvageable with median follow-up of 19 months (4-58-mos). Patients with failure were more likely to have diabetes mellitus, longer length of stay, higher American Society of Anesthesiologists (ASA) score, a stent placed at the time of pyeloplasty, or ureteral stent malfunction (P30-kg/m2 (P2 were associated with failure (P<0.05) while periureteral fibrosis trended toward a significant association (P=0.061). Conclusion: Nearly half of failures after LP are salvageable, many with endoscopic management.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90445/1/end-2E2010-2E0647.pd