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Rapid analysis of Forster resonance energy transfer by two-color global fluorescence correlation spectroscopy: trypsin proteinase reaction.

In this study we introduce the combination of two-color global fluorescence correlation spectroscopy (2CG-FCS) and Förster resonance energy transfer (FRET) as a very powerful combination for monitoring biochemical reactions on the basis of single molecule events. 2CG-FCS, which is a new variation emerging from the family of fluorescence correlation spectroscopy, globally analyzes the simultaneously recorded auto- and cross-correlation data from two photon detectors monitoring the fluorescence emission of different colors. Overcoming the limitations inherent in mere auto- and cross-correlation analysis, 2CG-FCS is sensitive in resolving and quantifying fluorescent species that differ in their diffusion characteristics and/or their molecular brightness either in one or both detection channels. It is able to account for effects that have often been considered as sources of severe artifacts in two-color and FRET measurements, the most prominent artifacts comprising photobleaching, cross talk, or concentration variations in sample preparation. Because of its very high statistical accuracy, the combination of FRET and 2CG-FCS is suited for high-throughput applications such as drug screening. Employing beam scanning during data acquisition even further enhances this capability and allows measurement times of <2 s. The improved performance in monitoring a FRET sample was verified by following the protease cleavage reaction of a FRET-active peptide. The FRET-inactive subpopulation of uncleaved substrate could be correctly assigned, revealing a substantial portion of inactive or missing acceptor label. The results were compared to those obtained by two-dimensional fluorescence intensity distribution analysis.

Biophysics↗

Umbilical vein white blood cell count as a marker of acidemia in term neonates.

OBJECTIVE: White blood cells are mobilized under both hypoxic and infectious conditions. Intrauterine hypoxia is linked to increased risk of cerebral palsy and is potentiated by the presence of infection. We hypothesized that umbilical vein white blood cell elevation in term neonates is associated with intrauterine acidemia. METHODS: We prospectively evaluated all liveborn neonates delivered at our institution for a 6-month period. Umbilical arterial blood was analyzed for pH and blood gas and venous blood for hematologic indices. Medical records of cases greater than or equal to 37 weeks' gestation were reviewed for correlative data. Student's t-test was used to determine difference of means and Chi-square test for goodness of fit. Pearson coefficients of correlation were applied where appropriate. RESULTS: A total of 1,948 liveborn, term neonates were delivered during the study period; 1,561 cases had white blood cell analysis and arterial blood gas data available. Acidemic cases had higher white blood cell (15.0 vs. 12.4 cells x 10(3)/mm3, P < 0.001), lymphocyte (4.43 vs. 3.59 cells X 10(3)/mm3, P < 0.0001), and neutrophil counts (9.08 vs. 7.71 cells x 10(3)/mm3, P < 0.01). As umbilical artery pH decreased, white blood cells became more prevalent. Likewise, as base deficit deepened, white blood cell counts increased. CONCLUSIONS: This study demonstrates an association between deepening acidemia and increasing white blood cell, lymphocyte, and neutrophil counts. Although statistically different, mean white blood cell counts for acidemic and nonacidemic cases are fairly close, limiting the clinical applicability in determining whether pathology is present in an individual case.

Acidosis↗

Phylogenetic analysis of hepatitis C virus isolates and their correlation to viremia, liver function tests, and histology.

Nucleotide sequence analysis of hepatitis C virus (HCV) strains showed substantial variability leading to a classification into several genotypes and subtypes. The data correlating HCV genotypes and subtypes with hepatitis C viremia levels, demographic characteristics of patients (age, mode of transmission, duration of infection), and severity of liver disease are conflicting. The interpretation of several studies is further complicated because the molecular methods used lacked specificity for genotyping/subtyping and underestimated viremia levels, especially in patients infected with HCV genotypes 2 and 3. In the present study we investigated 97 consecutive patients with chronic hepatitis C using molecular "gold standard" methods. HCV subtyping was performed by sequence and phylogenetic analysis of the nonstructural (NS)-5 region and serum HCV-RNA concentration was assessed by a validated genotype-independent quantitative reverse-transcription-polymerase chain reaction assay using an internal RNA standard. Patients infected with subtypes HCV-1b, HCV-2a-c, and HCV-4 were older than patients infected with HCV-1a and HCV-3a. Serum HCV-RNA levels ranged from 1.5 x 10(4) to 1.0 x 10(8) copies/mL with no significant differences between median serum HCV-RNA concentrations in patients infected with different genotypes/subtypes. Although patients infected with HCV-1b were older, no biochemical or histological evidence was obtained that this subtype is associated with more severe liver disease. Furthermore, the present study showed a lack of correlation between the serum HCV-RNA concentration, biochemical parameters, and liver histology. The median serum HCV-RNA levels in patients with chronic persistent hepatitis, chronic active hepatitis, and liver cirrhosis were 5.0 x 10(6) copies/mL, 2.5 x 10(6) copies/mL, and 5.0 x 10(6) copies/mL, respectively. These differences were not significant. In conclusion, using optimized and validated molecular techniques, the present cross-sectional study showed no correlation between HCV genotypes/subtypes, viremia, liver function test results, and histology.

Adult↗

Effect of multi-adhesive layering on retention of extraoral maxillofacial silicone prostheses in vivo.

STATEMENT OF PROBLEM: Loss of retention of maxillofacial prostheses often makes the margin visible or the prosthesis dislodge. Using several medical adhesives in combination may improve retention. PURPOSE: The purpose of this study was to investigate the effect of single- and multi-adhesive layering of 2 adhesives on the retention of maxillofacial silicone elastomer strips adhered to the skin of human forearms using a peel test. MATERIAL AND METHODS: Power analysis from a previous study and a pilot trial specified at least 20 subjects. Eight Silastic Adhesive A/MDX4-4210 silicone rubber strips (N=240) were applied in a predetermined random order to the left and right ventral forearms of 30 IRB-approved human subjects. Skin-Prep Protective Dressing was applied. Secure 2 Medical Adhesive (SMA) and Epithane-3 (E3) adhesive were used alone or as SMA/E3 or E3/SMA sandwiches (from skin to prosthesis) to adhere strips. Strips were peeled 6 hours later in a universal testing machine at 10 cm/min and data reported in N/m. Paired t tests were used to evaluate left and right arm differences. A Friedman test for nonparametric correlated data with within-subject design was performed, determining differences between both adhesives singly and in combination (alpha=.05). RESULTS: Tests of left-right differences were insignificant ( P =0.43), so the data from both arms were combined. Many strips with E3 did not adhere before testing and were counted as 0 adhesion. Median peel strengths (and 25th and 75th percentiles) in N/m were: SMA = 76.1 (47.1-107), E3 = 6.75 (0.0-25.9), SMA/E3 = 107 (78.0-132), and E3/SMA= 19.6 (6.99-42.4). All 4 variables were significantly different ( P <.0005). CONCLUSION: The multi-adhesive combination of SMA/E3 had the highest adhesion, followed, in order, by SMA alone, E3/ SMA, and E3 alone. Both E3 groups left a difficult-to-remove residue on the skin. SMA/E3 left a halo-like residue on the skin at the periphery of the strips from the E3 leaking around the SMA. SMA remained adherent to the prosthetic material.

Adhesives↗

Delays in implementing admission orders for critical care patients associated with length of stay in emergency departments in six mid-Atlantic states.

INTRODUCTION: Every day many admitted patients wait in emergency departments for available beds or for a receiving nurse to accomplish a transfer to an inpatient bed. The purpose of this study was to examine critical care patients' length of stay and time held in the emergency department once admitted to determine if (1) holding critical care patients in emergency departments after admission was related to skilled nursing shortages and/or limitations in available resources and (2) admission orders or tests may have been overlooked during this time. Little or no literature exists on this topic. METHODS: A Likert scale survey designed to yield descriptive comparative correlational data was sent to directors of critical care and emergency service areas. RESULTS: Received responses totaled 109. There is a positive correlation between increased length of stay and delays in implementation of admission orders while in emergency departments and tests missed or delayed upon arrival at the critical care unit. A majority of respondents indicated that ED nursing staff had responsibility for critical care admitted patients and other patients. Few indicated a formal process or committee was in place to address this issue specific to critical care patients. Limitations included a convenience sample and variations in operations related to size, location, and culture. DISCUSSION: Further study is necessary to determine whether patients' length of stay in the hospital is increased because of delays in plans of care and if patient outcomes are ultimately affected.

Bed Occupancy↗

Effect of previously failed kidney transplantation on peritoneal dialysis outcomes in the Australian and New Zealand patient populations.

BACKGROUND: There is limited information about the outcomes of patients commencing peritoneal dialysis (PD) after failed kidney transplantation. The aim of the present study was to compare patient survival, death-censored technique survival and peritonitis-free survival between patients initiating PD after failed renal allografts and those after failed native kidneys. METHODS: The study included all patients from the ANZDATA Registry who started PD between April 1, 1991 and March 31, 2004. Times to death, death-censored technique failure and first peritonitis episode were examined by multivariate Cox proportional hazards models. For all outcomes, conditional risk set models were utilized for the multiple failure data, and analyses were stratified by failure order. Standard errors were calculated by using robust variance estimation for the cluster-correlated data. RESULTS: In total, 13,947 episodes of PD were recorded in 23,579 person-years. Of these, 309 PD episodes were started after allograft failure. Compared with PD patients who had never undergone kidney transplantation, those with failed renal allografts were more likely to be younger, Caucasian, New Zealand residents and life-long non-smokers with lower body mass index (BMI), poorer initial renal function and a longer period from commencement of the first renal replacement therapy to PD. On multivariate analysis, PD patients with failed kidney transplants had comparable patient mortality [weighted hazards ratio (HR) 1.09, 95% confidence interval (CI) 0.81-1.45, P = 0.582], death-censored technique failure (adjusted HR 0.91, 95% CI 0.75-1.10, P = 0.315) and peritonitis-free survival (adjusted HR 0.92, 95% CI 0.72-1.16, P = 0.444) with those PD patients who had failed native kidneys. Similar findings were observed in a subset of patients (n = 5496) for whom peritoneal transport status was known and included in the models as a covariate. CONCLUSION: Patients commencing PD after renal allograft failure experienced outcomes comparable with those with failed native kidneys. PD appears to be a viable option for patients with failed kidney allografts.

Adult↗

Thioredoxin reductase regulates angiogenesis by increasing endothelial cell-derived vascular endothelial growth factor.

Low selenium (Se) status increases angiogenesis by inducing the production of vascular endothelial growth factor (VEGF); however, the mechanism responsible for VEGF up-regulation has yet to be characterized. Se's ability to control cellular oxidative state through its incorporation into selenoproteins such as thioredoxin reductase (TrxR) may explain previous studies that connect Se status to tumor angiogenesis. Therefore, the focus of this study was to determine if altered VEGF expression and angiogenesis due to decreased Se levels are influenced by reduced TrxR activity. We found that chemical inhibition of TrxR in Se-sufficient endothelial cells (ECs) was associated with increases in VEGF and VEGF receptor expression, cell migration, proliferation, and angiogenesis to levels similar to those seen in Se-deficient ECs. Specific inhibition of glutathione peroxidase did not affect pro-angiogenic responses, indicating a unique role of the TrxR system during low Se status. These data correlate changes in TrxR activity with changes in VEGF expression and angiogenic development in ECs, which is significant because minimal mechanistic data exist that explain the role of Se in cancer prevention. Understanding the importance of the tumor microenvironment in contributing to angiogenic regulation has the potential to significantly impact breast cancer chemoprevention strategies by focusing on maintaining proper EC function within the mammary gland.

Animals↗

Framework for application of geographic information system to the monitoring of dengue vectors.

In a successful management program of dengue vectors, not only health education, source reduction or insecticide application should be conducted, but all basic information should also be manipulated properly and efficiently. This information includes the surveys of species, dispersal and dynamics of vectors, as well as the detection of breeding sources, and the records of dengue cases and epidemic periods. Most of the above information expressed as regionalized variables always varies spatially and/or temporally. However, due to the deficiency of topological information, the conventional database management system cannot efficiently analyze those dengue related data. Thus, we have applied the geographic information system (GIS) to the monitoring of dengue vectors. The purpose of this report is to introduce the basic concepts of GIS, to describe the framework of the prototype dengue vector monitoring system which was built using data collected from the Sanmin area, Kaoshiung city, Taiwan, and to indicate the possibility of using this system to manipulate spatially correlated data and support decision making in the control of dengue disease.

Animals↗

Tobacco etch virus mRNA preferentially binds wheat germ eukaryotic initiation factor (eIF) 4G rather than eIFiso4G.

The 5'-leader of tobacco etch virus (TEV) genomic RNA directs the efficient translation from the naturally uncapped viral RNA. The TEV 143-nt 5'-leader folds into a structure that contains two domains, each of which contains RNA pseudoknots. The 5'-proximal pseudoknot 1 (PK1) is necessary to promote cap-independent translation (Zeenko, V., and Gallie, D. R. (2005) J. Biol. Chem. 280, 26813-26824). During the translation initiation of cellular mRNAs, eIF4G functions as an adapter that recruits many of the factors involved in stimulating 40 S ribosomal subunit binding to an mRNA. Two related but highly distinct eIF4G proteins are expressed in plants, animals, and yeast. The two plant eIF4G isoforms, referred to as eIF4G and eIFiso4G, differ in size (165 and 86 kDa, respectively) and their functional differences are still unclear. Although eIF4G is required for the translation of TEV mRNA, it is not known if eIF4G binds directly to the TEV RNA itself or if other factors are required. To determine whether binding affinity and isoform preference correlates with translational efficiency, fluorescence spectroscopy was used to measure the binding of eIF4G, eIFiso4G, and their complexes (eIF4F and eIFiso4F, respectively) to the TEV 143-nt 5'-leader (TEV1-143) and a shorter RNA that contained PK1. A mutant (i.e. S1-3) in which the stem of PK1 was disrupted resulting in impaired cap-independent translation, was also tested. These studies demonstrate that eIF4G binds TEV1-143 and PK1 RNA with approximately 22-30-fold stronger affinity than eIFiso4G. eIF4G and eIF4F bind TEV1-143 with similar affinity, whereas eIFiso4F binds with approximately 6-fold higher affinity than eIFiso4G. The binding affinity of eIF4G, eIF4F, and eIFiso4G to S1-3 was reduced by 3-5-fold, consistent with the reduction in the ability of this mutant to promote cap-independent translation. Temperature-dependent binding studies revealed that binding of the TEV 5'-leader to these initiation factors has a large entropic contribution. Overall, these results demonstrate the first direct interaction of eIF4G with the TEV 5'-leader in the absence of other initiation factors. These data correlate well with the observed translational data and provide more detailed information on the translational strategy of potyviruses.

Base Sequence↗

Tissue segmentation and classification of MRSI data using canonical correlation analysis.

In this article an accurate and efficient technique for tissue typing is presented. The proposed technique is based on Canonical Correlation Analysis, a statistical method able to simultaneously exploit the spectral and spatial information characterizing the Magnetic Resonance Spectroscopic Imaging (MRSI) data. Recently, Canonical Correlation Analysis has been successfully applied to other types of biomedical data, such as functional MRI data. Here, Canonical Correlation Analysis is adapted for MRSI data processing in order to retrieve in an accurate and efficient way the possible tissue types that characterize the organ under investigation. The potential and limitations of the new technique have been investigated by using simulated as well as in vivo prostate MRSI data, and extensive studies demonstrate a high accuracy, robustness, and efficiency. Moreover, the performance of Canonical Correlation Analysis has been compared to that of ordinary correlation analysis. The test results show that Canonical Correlation Analysis performs best in terms of accuracy and robustness.

Algorithms↗

The relationship between rhythmic activities during a mental task and sleep spindles: a correlative analysis.

In a previous study, we suggested that the characteristics of theta, alpha, and beta rhythms during a mental task were similar to those during sleep. Building upon the previous data, correlations between rhythmic activities during a mental task and during sleep were investigated in the present study. Patterns of correlation and no correlation between rhythmic activities during the mental task were similar to those during sleep for subjects with and without frontal midline theta (Fmtheta) activity. In the Fmtheta group, there were no correlations between rhythmic activities in the two situations, while in the non-Fmtheta subjects, theta and alpha rhythms showed a positive correlation with one another, and theta and beta rhythms correlated negatively during sleep. In both groups, there were many correlations between rhythmic activities during the mental task and those in Sleep Stage 2, while there were few correlations between rhythmic activities during the mental task and those in other sleep stages. These results suggest that the mechanism generating rhythmic activities during the appearance of rhythmic activities induced by a mental task may be closely related to those of rhythmic activities during sleep, and that the membrane potentials in reticular thalamic (RE) neurons during the appearance of rhythmic activities induced by a mental task may be nearly equivalent to that in Sleep Stage 2, and that the correlation pattern between the rhythmic activities in each group may be well explained by the appearance pattern of each rhythm in the previous report.

Adult↗

The relationship between angiogenesis and cyclooxygenase-2 expression in prostate cancer.

OBJECTIVE: To test the hypothesis that angiogenesis in prostate cancer is associated with tumour invasion and metastasis, and that this is mediated through increased cyclooxygenase-2 (COX-2) expression. PATIENTS AND METHODS: Angiogenesis was assessed in 105 patients with either prostate cancer (79) or benign prostatic hyperplasia (BPH, 26) and these data correlated with levels of COX-2 expression in the same dataset. The mean microvessel density (MVD) was analysed as a marker of angiogenesis, using the endothelial antigen CD34 stained by immunohistochemistry. RESULTS: There was no difference in MVD in progressive tumour stages compared with BPH. There was a negative correlation between MVD and COX-2 expression, but the effect of increased COX-2 expression on MVD was not marked. CONCLUSION: These data suggest that COX-2 drives tumour spread in prostate cancer by means other than the promotion of angiogenesis.

Aged↗

A comparison of medical record with billing diagnostic information associated with ambulatory medical care.

The degree of similarity between diagnostic information furnished with claims and that simultaneously entered into the medical record was estimated for 1,215 private office visits in British Columbia, Canada. For each visit, claim card and chart diagnoses were compared by having three independent internists (blinded to source and type of the data) make judgments about each diagnostic pair. The judges were highly consistent internally and their judgments were stable over time. In 40 per cent of cases chart and claims data were judged dissimilar, and in 38 per cent of cases claims data were judged more valuable as a reflection of the primary problem treated. The degree of judged similarity of chart and claims data correlated significantly and negatively with physician workload, income, and judges' preference for the billing card diagnosis. We conclude that in using claims data to determine the content of ambulatory visits, independent validation of such data may be important.

Ambulatory Care↗

[Methodology of phase II clinical trials of hypocholesteremic drugs: which criteria?].

Phase II is crucial in drug trials: during this period the first patients are exposed to the drug and the results influence the continuation of the development and the success of later trials. This stage is fundamental in lipid lowering drugs considering the costs of development and potential benefits for public health. The choice of pertinent criteria for the design of a lipid lowering drug trial depends on the analysis of data acquired during previous large scale trials. These criteria should demonstrate the potential of the tested drug and the best conditions of its administration. It is especially at this stage that the drug dosage optimising the efficacy/tolerance ratio should be determined. Analysis of large scale prevention trials indicates LDL-cholesterol as the principal criterion. It is related to coronary risk in the major trials of lipid lowering drugs. A reduction of LDL-cholesterol gives a good numerical estimate of the expected reduction of cardiovascular risk. It is then possible to formulate the hypotheses required for the design of these trials, in particular to calculate the size of the treatment group required. The total cholesterol should also be measured above all because of the large amount of data correlating it with cardiovascular risk. The HDL-cholesterol and triglycerides should also be taken into account even though some of the data concerning these parameters is controversial. The discrimination obtained by measuring HDL-cholesterol is without any doubt crude, and future criteria should enable more accurate and rapid evaluation of lipid lowering activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Anticholesteremic Agents↗

The relationship between class I binding affinity and immunogenicity of potential cytotoxic T cell epitopes.

The relationship between binding affinity for HLA class I molecules and immunogenicity of discrete peptide epitopes has been analyzed in two different experimental approaches. In the first approach, the immunogenicity of potential epitopes ranging in MHC binding affinity over a 10,000-fold range was analyzed in HLA-A*0201 transgenic mice. In the second approach, the antigenicity of approximately 100 different hepatitis B virus (HBV)-derived potential epitopes, all carrying A*0201 binding motifs, was assessed by using PBL of acute hepatitis patients. In both cases, it was found that an affinity threshold of approximately 500 nM (preferably 50 nM or less) apparently determines the capacity of a peptide epitope to elicit a CTL response. These data correlate well with class I binding affinity measurements of either naturally processed peptides or previously described T cell epitopes. Taken together, these data have important implications for the selection of epitopes for peptide-based vaccines, and also formally demonstrate the crucial role of determinant selection in the shaping of T cell responses. Because in most (but not all) cases, high affinity peptides seem to be immunogenic, our data also suggest that holes in the functional T cell repertoire, if they exist, may be relatively rare.

Amino Acid Sequence↗

A semi-parametric Bayesian approach to generalized linear mixed models.

The linear mixed effects model with normal errors is a popular model for the analysis of repeated measures and longitudinal data. The generalized linear model is useful for data that have non-normal errors but where the errors are uncorrelated. A descendant of these two models generates a model for correlated data with non-normal errors, called the generalized linear mixed model (GLMM). Frequentist attempts to fit these models generally rely on approximate results and inference relies on asymptotic assumptions. Recent advances in computing technology have made Bayesian approaches to this class of models computationally feasible. Markov chain Monte Carlo methods can be used to obtain 'exact' inference for these models, as demonstrated by Zeger and Karim. In the linear or generalized linear mixed model, the random effects are typically taken to have a fully parametric distribution, such as the normal distribution. In this paper, we extend the GLMM by allowing the random effects to have a non-parametric prior distribution. We do this using a Dirichlet process prior for the general distribution of the random effects. The approach easily extends to more general population models. We perform computations for the models using the Gibbs sampler.

Bayes Theorem↗

Calcium, magnesium, copper and zinc content of menu components: comparison of analysed with calculated values.

The objective of this study was to show whether analysed nutrient data correlate with calculated values from a food database. For this purpose the calcium, magnesium, copper and zinc contents of 62 menu components from lunch menus were determined with ICP-AES. Then the analysed values were compared with calculated values from a widely used food database. Our results indicated that there is a significant correlation (p < 0.01) between the analysed and calculated values of all four elements. The correlation coefficient as determined with nonparametric correlation analysis was 0.807 for calcium, 0.786 for magnesium, 0.772 for zinc, and 0.414 for copper. Although these correlations are significant, great differences between analysed and calculated values for all four elements could be found when considering single menu components. Therefore data of studies illustrating analysed with calculated values of single menu components have practical importance.

Algorithms↗

Stimulus dependent neural correlation: an example from the cochlear nucleus.

Results of several recent cross-correlation studies have been interpreted in terms of "neuronal plasticity" and "stimulus dependent wiring diagrams" produced by presumed dynamic neural reorganization mechanisms. Presented here are examples of stimulus-dependent cross-correlograms observed in a pair of type IV units recorded in the dorsal cochlear nucleus (DCN). The interpretation of these correlation data is based on current hypotheses of DCN circuitry. It is suggested that plasticity mechanisms are not responsible for these stimulus-dependent correlations. A more likely mechanism is one that allows various portions of a hard wired neural circuit to be selectively activated by the stimuli.

Acoustic Stimulation↗