Correction of timing errors in photomultiplier tubes used in phase-modulation fluorometry

The measurement of fluorescence lifetimes is known to be hindered by the wavelenght-dependent and photocathode area-dependent time response of photomultiplier tubes. A simple and direct method is described to minimize the effects in photomultiplier tubes for phase-modulation fluorometry. Reference fluorophores of known lifetime were used in place of the usual scattering reference. The emission wavelenghts of the reference and sample were matched by either filters or a monochromator, and the use of a fluorophore rather than a scatter decreases the differences in spatial distribution of light emanating from the reference and sample. Thus photomultiplier tube artifacts are minimized. Five reference fluorophores were selected on the basis of availability, ease of solution preparation, and constancy of lifetime with temperature and emission wavelenght. These compounds are p-terphenyl, PPO, PPD, POPOP and dimethyl POPOP. These compounds are dissolved in ethanol to give standard solutions that can be used over the temperature range from ?55 to +55°C. Purging with inert gas is not necessary. The measured phase and modulation of the reference solution is used, in conjunction with the known reference, lifetime, to calculate the actual phase and modulation of the exictation beam. The use of standard fluorophores does not require separate experiments to quantify photomultiplier effects, and does not increase the time required for the measurement of fluorescence lifetimes. Examples are presented which demonstrate the elimination of artifactual photomultiplier effects in measurements of the lifetimes of DADH (0.4 ns) and indole solutions quenched by iodide. In addition, the use of these reference solutions increases the accuracy of fluorescence lifetime measurements ranging ranging to 30 ns. We judge this method to provide more reliable lifetime measurements by the phase and modulation method. The test solutions and procedures we describe may be used by other laboratories to evaluate the performance of their phase fluorometers.     

Stereotactic Body Radiation Therapy for Primary and Metastatic Liver Tumors

OBJECTIVES: The full potential of stereotactic body radiation therapy (SBRT), in the treatment of unresectable intrahepatic malignancies, has yet to be realized as our experience is still limited. Thus, we evaluated SBRT outcomes for primary and metastatic liver tumors, with the goal of identifying factors that may aid in optimization of therapy. METHODS: From2005 to 2010, 62 patients with 106 primary and metastatic liver tumors were treated with SBRT to a median biologic effective dose (BED) of 100 Gy (42.6-180). The majority of patients received either three (47%) or five fractions (48%). Median gross tumor volume (GTV) was 8.8 cm³ (0.2-222.4). RESULTS: With a median followup of 18 months (0.46-46.8), freedom from local progression (FFLP) was observed in 97 of 106 treated tumors, with 1- and 2-year FFLP rates of 93% and 82%. Median overall survival (OS) for all patients was 25.2 months, with 1- and 2-year OS of 81%and 52%. Neither BED nor GTV significantly predicted for FFLP. Local failure was associated with a higher risk of death [hazard ratio (HR) = 5.1, P = .0007]. One Child-Pugh Class B patient developed radiationinduced liver disease. There were no other significant toxicities. CONCLUSIONS: SBRT provides excellent local control for both primary and metastatic liver lesions with minimal toxicity. Future studies should focus on appropriate selection of patients and on careful assessment of liver function to maximize both the safety and efficacy of treatment.     

Variations in enzyme activity in stomach and pancreatic tissue and digesta in piglets around weaning

A study was performed to investigate the effect of weaning at 4 weeks of age on the activity of digestive enzymes in the stomach and pancreatic tissue and in digesta from 3 days prior to weaning to 9 days postweaning in 64 piglets. In stomach tissue the activity of pepsin and gastric lipase was determined. Pepsin activity declined abruptly after weaning but 5 days postweaning the weaning level was regained and in the gastric contents no change in pepsin activity was observed. Weaning did not influence the activity of gastric lipase. The activity of eight enzymes and a cofactor was measured in pancreatic tissue. The effect of weaning on the enzyme activity was highly significant for all enzymes except elastase. The activity of all enzymes remained at the weaning level during day 1–2 postweaning followed by a reduction of the activity. The activity of trypsin, carboxypeptidase A, amylase and lipase exhibited minimum activity 5 days postweaning. Trypsin activity increased to the preweaning level on day 7–9 whereas the activity of the others increased but did not reach the preweaning level. The activity of chymotrypsin, carboxypeptidase B and carboxyl ester hydrolase decreased during the entire experimental period. In digesta no effect of weaning was observed on the activity of amylase and trypsin. The activity of chymotrypsin was reduced after weaning in the proximal third of the small intestine and lipase and carboxyl ester hydrolase activity was reduced in the middle and distal parts of the small intestine after weaning. The present study shows that the activities of the digestive enzymes in the pancreatic tissue are affected by weaning. Even though the pancreatic secretion cannot be judged from these results they show that the enzymes respond differently to weaning. In general the activity of the digestive enzymes in pancreatic tissue is low on day 5 postweaning which in interaction with other factors may increase the risk of developing postweaning diarrhoea.     

A knowledge-driven agent-centred framework for data mining in EMG

In this paper, we present a multi-agent framework for data mining in electromyography. This application, based on a web interface, provides a set of functionalities allowing to manipulate 1000 medical cases and more than 25,000 neurological tests stored in a medical database. The aim is to extract medical information using data mining algorithms and to supply a knowledge base with pertinent information. The multi-agent platform gives the possibility to distribute the data management process between several autonomous entities. This framework provides a parallel and flexible data manipulation.     

The ITGAV rs3738919 variant and susceptibility to rheumatoid arthritis in four Caucasian sample sets

Introduction

Angiogenesis is an important process in the development of destructive synovial pannus in rheumatoid arthritis (RA). The ITGAV +gene encodes a cell cycle-associated antigen, integrin ανβ 3, which plays a role in RA angiogenesis. Previously, two independent studies identified an association between the major allele of the ITGAV single-nucleotide polymorphism (SNP) rs3738919 and RA. We therefore tested this association in an independent study using New Zealand (NZ) and Oxford (UK) RA case control samples.

Methods

We compared genotype frequencies in 740 NZ Caucasian RA patients and 553 controls genotyped for rs3738919, using a polymerase chain reaction-restriction fragment length polymorphism assay. A TaqMan genotyping SNP assay was used to type 713 Caucasian RA patients and 515 control samples from Oxford for the rs3738919 variant. Association of rs3738919 with RA was tested in these two sample sets using the chi-square goodness-of-fit test. The Mantel-Haenszel test was used to perform a meta-analysis, combining the genetic results from four independent Caucasian case control cohorts, consisting of 3,527 cases and 4,126 controls. Haplotype analysis was also performed using SNPs rs3911238, rs10174098 and rs3738919 in the Wellcome Trust Case Control Consortium, NZ and Oxford case control samples.

Results

We found no evidence for association between ITGAV and RA in either the NZ or Oxford sample set (odds ratio [OR] = 0.88, P_allelic = 0.11 and OR = 1.18, P_allelic = 0.07, respectively). Inclusion of these data in a meta-analysis (random effects) of four independent cohorts (3,527 cases and 4,126 controls) weakens support for the hypothesis that rs3738919 plays a role in the development of RA (OR_combined = 0.92, 95% confidence interval 0.80 to 1.07; P = 0.29). No consistent haplotype associations were evident.

Conclusions

Association of ITGAV SNP rs7378919 with RA was not replicated in NZ or Oxford case control sample sets. Meta-analysis of these and previously published data lends limited support for a role for the ITGAV in RA in Caucasians of European ancestry.     

Rotational freedom of tryptophan residues in proteins and peptides

We studied the rotational motions of tryptophan residues in proteins and peptides by measurement of steady-state fluorescence anisotropies under conditions of oxygen quenching. By fluorescence quenching we can shorten the fluorescence lifetime and thereby decrease the average time for rotational diffusion prior to fluorescence emission. This method allowed measurement of rotational correlation times ranging from 0.03 to 50 ns, when the unquenched fuorescence lifetimes are near 4 ns. A wide range of proteins and peptides were investigated with molecular weights ranging from 200 to 80 000. Many of the chosen substances possessed a single tryptophan residue to minimize the uncertainties arising from a heterogeneous population of fluorophores. In addition, we also studied a number of multi-tryptophan proteins. Proteins were studied at various temperatures, under conditions of self-association, and in the presence of denaturants. A wide variety of rotational correlation times were found. As examples we note that the single tryptophan residue of myelin basic protein was highly mobile relative to overall protein rotation whereas tryptophan residues in human serum albumin, RNase T1, aldolase, and horse liver alcohol dehydrogenase were found to be immobile relative to the protein matrix. These results indicate that one cannot generalize about the extent of segmental mobility of the tryptophan residues in proteins. This physical property of proteins is highly variable between proteins and probably between different regions of the same protein.     

Quantification of angiogenesis in estrogen receptor-positive and negative breast carcinoma

Background

The objective of this study was to evaluate angiogenesis according to CD34 antigen expression in estrogen receptor (ER)-positive and negative breast carcinomas.

Methods

This study comprised 64 cases of infiltrating ductal carcinoma in postmenopausal women divided into two groups: Group A: ER-positive, n = 35; and Group B: ER-negative, n = 29. The anti-CD34 monoclonal antibody was used as a marker for endothelial cells. Microvessel count was carried out in 10 fields per slide using a 40× objective lens (magnification 400×). Statistical analysis of the data was performed using Student's t-test (p < 0.05).

Results

The mean number of vessels stained with the anti-CD34 antibody in the estrogen receptor-positive and negative tumors was 23.51 ± 1.15 and 40.24 ± 0.42, respectively. The number of microvessels was significantly greater in the estrogen receptor-negative tumors (p < 0.001).

Conclusion

ER-negative tumors have significantly greater CD34 antigen expression compared to ER-positive tumors.

     

Sequence dependence of chromosomal R-loops at the immunoglobulin heavy-chain Smu class switch region

The mechanism by which the cytidine deaminase activation-induced deaminase (AID) acts at immunoglobulin heavy-chain class switch regions during mammalian class switch recombination (CSR) remains unclear. R-loops have been proposed as a basis for this targeting. Here, we show that the difference between various forms of the Smu locus that can or cannot undergo CSR correlates well with the locations and detectability of R-loops. The Smu R-loops can initiate hundreds of base pairs upstream of the core repeat switch regions, and the area where the R-loops initiate corresponds to the zone where the AID mutation frequency begins to rise, despite a constant density of WRC sites in this region. The frequency of R-loops is 1 in 25 alleles, regardless of the presence of the core Smu repeats, again consistent with the initiation of most R-loops upstream of the core repeats. These findings explain the surprisingly high levels of residual CSR in B cells from mice lacking the core Smu repeats but the marked reduction in CSR in mice with deletions of the region upstream of the core Smu repeats. These studies also provide the first analysis of how R-loop formation in the eukaryotic chromosome depends on the DNA sequence.