Alteration of RNA Splicing by Small-Molecule Inhibitors of the Interaction between NHP2L1 and U4.

Splicing is an important eukaryotic mechanism for expanding the transcriptome and proteome, influencing a number of biological processes. Understanding its regulation and identifying small molecules that modulate this process remain a challenge. We developed an assay based on time-resolved fluorescence resonance energy transfer (TR-FRET) to detect the interaction between the protein NHP2L1 and U4 RNA, which are two key components of the spliceosome. We used this assay to identify small molecules that interfere with this interaction in a high-throughput screening (HTS) campaign. Topotecan and other camptothecin derivatives were among the top hits. We confirmed that topotecan disrupts the interaction between NHP2L1 and U4 by binding to U4 and inhibits RNA splicing. Our data reveal new functions of known drugs that could facilitate the development of therapeutic strategies to modify splicing and alter gene function.

Structure and mechanism of the phage T4 recombination mediator protein UvsY.

The UvsY recombination mediator protein is critical for efficient homologous recombination in bacteriophage T4 and is the functional analog of the eukaryotic Rad52 protein. During T4 homologous recombination, the UvsX recombinase has to compete with the prebound gp32 single-stranded binding protein for DNA-binding sites and UvsY stimulates this filament nucleation event. We report here the crystal structure of UvsY in four similar open-barrel heptameric assemblies and provide structural and biophysical insights into its function. The UvsY heptamer was confirmed in solution by centrifugation and light scattering, and thermodynamic analyses revealed that the UvsY-ssDNA interaction occurs within the assembly via two distinct binding modes. Using surface plasmon resonance, we also examined the binding of UvsY to both ssDNA and the ssDNA-gp32 complex. These analyses confirmed that ssDNA can bind UvsY and gp32 independently and also as a ternary complex. They also showed that residues located on the rim of the heptamer are required for optimal binding to ssDNA, thus identifying the putative ssDNA-binding surface. We propose a model in which UvsY promotes a helical ssDNA conformation that disfavors the binding of gp32 and initiates the assembly of the ssDNA-UvsX filament.

Clinical predictors for laboratory-confirmed influenza infections: exploring case definitions for influenza-like illness.

OBJECTIVE: To identify clinical signs and symptoms (ie, "terms") that accurately predict laboratory-confirmed influenza cases and thereafter generate and evaluate various influenza-like illness (ILI) case definitions for detecting influenza. A secondary objective explored whether surveillance of data beyond the chief complaint improves the accuracy of predicting influenza. DESIGN: Retrospective, cross-sectional study. SETTING: Large urban academic medical center hospital. PARTICIPANTS: A total of 1,581 emergency department (ED) patients who received a nasopharyngeal swab followed by rRT-PCR testing between August 30, 2009, and January 2, 2010, and between November 28, 2010, and March 26, 2011. METHODS: An electronic surveillance system (GUARDIAN) scanned the entire electronic medical record (EMR) and identified cases containing 29 clinical terms relevant to influenza. Analyses were conducted using logistic regressions, diagnostic odds ratio (DOR), sensitivity, and specificity. RESULTS: The best predictive model for identifying influenza for all ages consisted of cough (DOR=5.87), fever (DOR=4.49), rhinorrhea (DOR=1.98), and myalgias (DOR=1.44). The 3 best case definitions that included combinations of some or all of these 4 symptoms had comparable performance (ie, sensitivity=89%-92% and specificity=38%-44%). For children <5 years of age, the addition of rhinorrhea to the fever and cough case definition achieved a better balance between sensitivity (85%) and specificity (47%). For the fever and cough ILI case definition, using the entire EMR, GUARDIAN identified 37.1% more influenza cases than it did using only the chief complaint data. CONCLUSIONS: A simplified case definition of fever and cough may be suitable for implementation for all ages, while inclusion of rhinorrhea may further improve influenza detection for the 0-4-year-old age group. Finally, ILI surveillance based on the entire EMR is recommended.

Comparing the accuracy of syndrome surveillance systems in detecting influenza-like illness: GUARDIAN vs. RODS vs. electronic medical record reports.

BACKGROUND: A highly sensitive real-time syndrome surveillance system is critical to detect, monitor, and control infectious disease outbreaks, such as influenza. Direct comparisons of diagnostic accuracy of various surveillance systems are scarce. OBJECTIVE: To statistically compare sensitivity and specificity of multiple proprietary and open source syndrome surveillance systems to detect influenza-like illness (ILI). METHODS: A retrospective, cross-sectional study was conducted utilizing data from 1122 patients seen during November 1­7, 2009 in the emergency department of a single urban academic medical center. The study compared the Geographic Utilization of Artificial Intelligence in Real-time for Disease Identification and Alert Notification (GUARDIAN) system to the Complaint Coder (CoCo) of the Real-time Outbreak Detection System (RODS), the Symptom Coder (SyCo) of RODS, and to a standardized report generated via a proprietary electronic medical record (EMR) system. Sensitivity, specificity, and accuracy of each classifier's ability to identify ILI cases were calculated and compared to a manual review by a board-certified emergency physician. Chi-square and McNemar's tests were used to evaluate the statistical difference between the various surveillance systems.ResultsThe performance of GUARDIAN in detecting ILI in terms of sensitivity, specificity, and accuracy, as compared to a physician chart review, was 95.5%, 97.6%, and 97.1%, respectively. The EMR-generated reports were the next best system at identifying disease activity with a sensitivity, specificity, and accuracy of 36.7%, 99.3%, and 83.2%, respectively. RODS (CoCo and SyCo) had similar sensitivity (35.3%) but slightly different specificity (CoCo = 98.9%; SyCo = 99.3%). The GUARDIAN surveillance system with its multiple data sources performed significantly better compared to CoCo (χ2 = 130.6, p < 0.05), SyCo (χ2 = 125.2, p < 0.05), and EMR-based reports (χ2 = 121.3, p < 0.05). In addition, similar significant improvements in the accuracy (>12%) and sensitivity (>47%) were observed for GUARDIAN with only chief complaint data as compared to RODS (CoCo and SyCo) and EMR-based reports. CONCLUSION: In our study population, the GUARDIAN surveillance system, with its ability to utilize multiple data sources from patient encounters and real-time automaticity, demonstrated a more robust performance when compared to standard EMR-based reports and the RODS systems in detecting ILI. More large-scale studies are needed to validate the study findings, and to compare the performance of GUARDIAN in detecting other infectious diseases.

The impact of alternative diagnoses on the utility of influenza-like illness case definition to detect the 2009 H1N1 pandemic.

OBJECTIVE: To investigate the impact of excluding cases with alternative diagnoses on the sensitivity and specificity of the Centers for Disease Control and Prevention's (CDC) influenza-like illness (ILI) case definition in detecting the 2009 H1N1 influenza, using Geographic Utilization of Artificial Intelligence in Real-Time for Disease Identification and Alert Notification, a disease surveillance system. DESIGN: Retrospective cross-sectional study design. SETTING: Emergency department of an urban tertiary care academic medical center. PATIENTS: 1,233 ED cases, which were tested for respiratory viruses from September 5, 2009 to May 5, 2010. MAIN OUTCOME MEASURE: The main outcome measures were positive predictive value, negative predictive value, sensitivity, specificity, and accuracy of the ILI case definition (both including and excluding alternative diagnoses) to detect H1N1. RESULTS: There was a significant decrease in sensitivity (chi2 = 9.09, p < 0.001) and significant improvement in specificity (chi2 = 179, p < 0.001), after excluding cases with alternative diagnoses. CONCLUSION: When early detection of an influenza epidemic is of prime importance, pursuing alternative diagnoses as part of CDC's ILI case definition may not be warranted for public health reporting due to the significant decrease in sensitivity, in addition to the resources required for detecting these alternative diagnoses.

Variable-density flow and transport simulation of wellbore brine displacement.

Borehole dilution tests have been used for characterization of aquifer hydrogeologic properties for several decades. Based on the principles of borehole dilution tests, we conducted what more appropriately may be considered a wellbore fluid displacement test in a limestone aquifer in South Carolina. Our study area is a quarry in the coastal plain of South Carolina. Using a solution of reagent grade NaCl and deionized H(2)O as a tracer, a brine slug was introduced into a 5 cm (2 in.) diameter Schedule 40 PVC well with a 6-m slotted screen at the bottom. Immediately following addition of the brine, a recording electrical conductivity (EC) sensor was placed in the well opposite the screen and set to record EC in 2-min intervals for 5 days. An alternative to previous methods for analyzing data from wellbore brine displacement tests was developed. Results were analyzed using SEAWAT-2000 to account for the density dependency of brine flow and transport. The high spatial resolution, three-dimensional numerical simulation enabled direct incorporation of well construction peculiarities, including the sand pack and length of screen, in the data analysis. Hydraulic conductivity, effective porosity, and longitudinal dispersivity were adjusted in the simulation model until the best match of simulated wellbore fluid concentrations to observed concentrations was achieved. Using this procedure, we were able to obtain a very close agreement between observed and simulated concentrations and, hence, reliable estimates of the hydrogeologic properties of the aquifer in the vicinity of the test well.

Antidiabetic therapy before and 1 year after discharge for patients manifesting in-hospital hyperglycemia.

BACKGROUND: While studies have evaluated the impact of hyperglycemia during hospitalization, little is known about its management before and after admission. METHODS: We sampled a managed care outpatient database (8547 patients) with linkage to inpatient data from June 1, 2003 to June 30, 2006, evaluating hyperglycemia management preadmission (PA), during index admission (IA), and postdischarge (PD). Antihyperglycemic medications used during PA, IA, and PD for up to 15 months were available for 2898 patients from this cohort. Diabetes mellitus (DM) status was determined from ICD-9 codes. RESULTS: Patients at IA had an average age of 60 +/- 18 years. Forty-one percent were men and 59% were women. Nearly 60% of patients either had DM or manifested hyperglycemia (blood glucose > 130 mg/dL): 19.5% (1627) had preexisting DM (DM+); 9.6% (801) were newly diagnosed with DM at IA; 28.6% (2391) did not have DM (DM-) but manifested hyperglycemia during hospitalization; and 36.9% (3083) remained normoglycemic. The DM status of 459 patients (5.4%) was unascertainable. For the previously diagnosed DM+ patients, antidiabetic therapy intensified more than 2-fold during IA, primarily with insulin. Postdischarge hyperglycemia management medication doubled, with increases seen in both oral hypoglycemic agents (OHAs) and insulin. Newly diagnosed DM+ patients were also treated primarily with insulin during hospitalization and reverted to OHAs PD. A minority of DM- patients received antidiabetic therapy during IA, primarily with insulin, but 4% were diagnosed DM+ in the 15-month period PD and treated primarily with OHAs. Among those normoglycemic in the hospital, 1% were diagnosed with DM and treated with OHAs PD. CONCLUSION: Hyperglycemia management was intensified for all DM+ patients, primarily with insulin in the hospital and both insulin and OHAs PD. A better understanding of this natural history and antidiabetic transitional care could facilitate better discharge planning and thus improve diabetes care.

Evaluation of multiple models to distinguish closely related forms of disease using DNA microarray data: an application to multiple myeloma.

MOTIVATION: Standard laboratory classification of the plasma cell dyscrasia monoclonal gammopathy of undetermined significance (MGUS) and the overt plasma cell neoplasm multiple myeloma (MM) is quite accurate, yet, for the most part, biologically uninformative. Most, if not all, cancers are caused by inherited or acquired genetic mutations that manifest themselves in altered gene expression patterns in the clonally related cancer cells. Microarray technology allows for qualitative and quantitative measurements of the expression levels of thousands of genes simultaneously, and it has now been used both to classify cancers that are morphologically indistinguishable and to predict response to therapy. It is anticipated that this information can also be used to develop molecular diagnostic models and to provide insight into mechanisms of disease progression, e.g., transition from healthy to benign hyperplasia or conversion of a benign hyperplasia to overt malignancy. However, standard data analysis techniques are not trivial to employ on these large data sets. Methodology designed to handle large data sets (or modified to do so) is needed to access the vital information contained in the genetic samples, which in turn can be used to develop more robust and accurate methods of clinical diagnostics and prognostics. RESULTS: Here we report on the application of a panel of statistical and data mining methodologies to classify groups of samples based on expression of 12,000 genes derived from a high density oligonucleotide microarray analysis of highly purified plasma cells from newly diagnosed MM, MGUS, and normal healthy donors. The three groups of samples are each tested against each other. The methods are found to be similar in their ability to predict group membership; all do quite well at predicting MM vs. normal and MGUS vs. normal. However, no method appears to be able to distinguish explicitly the genetic mechanisms between MM and MGUS. We believe this might be due to the lack of genetic differences between these two conditions, and may not be due to the failure of the models. We report the prediction errors for each of the models and each of the methods. Additionally, we report ROC curves for the results on group prediction. AVAILABILITY: Logistic regression: standard software, available, for example in SAS. Decision trees and boosted trees: C5.0 from www.rulequest.com. SVM: SVM-light is publicly available from svmlight.joachims.org. Naïve Bayes and ensemble of voters are publicly available from www.biostat.wisc.edu/~mwaddell/eov.html. Nearest Shrunken Centroids is publicly available from http://www-stat.stanford.edu/~tibs/PAM.

Collagen stability: insights from NMR spectroscopic and hybrid density functional computational investigations of the effect of electronegative substituents on prolyl ring conformations.

Collagen-like peptides of the type (Pro-Pro-Gly)(10) fold into stable triple helices. An electron-withdrawing substituent at the H(gamma)(3) ring position of the second proline residue stabilizes these triple helices. The aim of this study was to reveal the structural and energetic origins of this effect. The approach was to obtain experimental NMR data on model systems and to use these results to validate computational chemical analyses of these systems. The most striking effects of an electron-withdrawing substituent are on the ring pucker of the substituted proline (Pro(i)) and on the trans/cis ratio of the Xaa(i-1)-Pro(i) peptide bond. NMR experiments demonstrated that N-acetylproline methyl ester (AcProOMe) exists in both the C(gamma)-endo and C(gamma)-exo conformations (with the endo conformation slightly preferred), N-acetyl-4(R)-fluoroproline methyl ester (Ac-4R-FlpOMe) exists almost exclusively in the C(gamma)-exo conformation, and N-acetyl-4(S)-fluoroproline methyl ester (Ac-4S-FlpOMe) exists almost exclusively in the C(gamma)-endo conformation. In dioxane, the K(trans/cis) values for AcProOMe, Ac-4R-FlpOMe, and Ac-4S-FlpOMe are 3.0, 4.0, and 1.2, respectively. Density functional theory (DFT) calculations with the (hybrid) B3LYP method were in good agreement with the experimental data. Computational analysis with the natural bond orbital (NBO) paradigm shows that the pucker preference of the substituted prolyl ring is due to the gauche effect. The backbone torsional angles, phi and psi, were shown to correlate with ring pucker, which in turn correlates with the known phi and psi angles in collagen-like peptides. The difference in K(trans/cis) between AcProOMe and Ac-4R-FlpOMe is due to an n-->pi interaction associated with the Bürg-Dunitz trajectory. The decrease in K(trans/cis) for Ac-4S-FlpOMe can be explained by destabilization of the trans isomer because of unfavorable electronic and steric interactions. Analysis of the results herein along with the structures of collagen-like peptides has led to a theory that links collagen stability to the interplay between the pyrrolidine ring pucker, phi and psi torsional angles, and peptide bond trans/cis ratio of substituted proline residues.