Mannan-binding lectin MBL2 gene polymorphism in chronic hepatitis C: association with the severity of liver fibrosis and response to interferon therapy

Hepatitis C virus (HCV) is a major cause of hepatic disease and of liver transplantation worldwide. Mannan-binding lectin (MBL), encoded by the MBL2 gene, can have an important role as an opsonin and complement activating molecule in HCV persistence and liver injury. We assessed the MBL2 polymorphism in 102 Euro-Brazilian patients with moderate and severe chronic hepatitis C, paired for gender and age with 102 HCV seronegative healthy individuals. Six common single nucleotide polymorphisms in the MBL2 gene, three in the promoter (H/L, X/Y and P/Q) and three in exon 1 (A, the wild-type, and B, C or D also known as O) were evaluated using real-time polymerase chain reaction with fluorescent hybridization probes. The concentration of MBL in plasma was measured by enzyme-linked immunosorbent assay. The frequency of the YA/YO genotype was significantly higher in the HCV patients compared with the controls (P = 0.022). On the other hand, the genotypes associated with low levels of MBL (XA/XA, XA/YO and YO/YO) were decreased significantly in the patients with severe fibrosis (stage F4), when compared with the patients with moderate fibrosis (stage F2) (P = 0.04) and to the control group (P = 0.011). Furthermore, MBL2 genotypes containing X or O mutations were found to be associated with non-responsiveness to pginterferon and ribavirin treatment (P = 0.023). MBL2 polymorphisms may therefore be associated not only with the development of chronic hepatitis C, but also with its clinical evolution and response to treatment.

Calculating current densities and fields produced by shielded magnetic resonance imaging probes

A method is presented for computing the fields produced by radio frequency probes of the type used in magnetic resonance imaging. The effects of surrounding the probe with a shielding coil, intended to eliminate stray fields produced outside the probe, are included. An essential feature of these devices is the fact that the conducting rungs of the probe are of finite width relative to the coil radius, and it is therefore necessary to find the distribution of current within the conductors as part of the solution process. This is done here using a numerical method based on the inverse finite Hilbert transform, applied iteratively to the entire structure including its shielding coils. It is observed that the fields are influenced substantially by the width of the conducting rungs of the probe, since induced eddy currents within the rungs become more pronounced as their width is increased. The shield is also shown to have a significant effect on both the primary current density and the resultant fields. Quality factors are computed for these probes and compared with values measured experimentally.

A novel method for development of species and strain-specific DNA probes and PCR primers for identifying Burkholderia solanacearum (formerly Pseudomonas solanacearum)

Six Burkholderia solanacearum (formerly Pseudomonas solanacearum) genomic DNA fragments were isolated, using RAPD techniques and cloning, from the three genetically diverse strains: ACH092 (Biovar 4), ACH0158 (Biovar 2) and ACH0171 (Biovar 3) (1). One of these cloned fragments was selected because it was present constantly in all bacterial strains analysed. The remaining five clones were selected because Southern hybridisation revealed that each showed partial or complete specificity towards the strain of origin. A seventh genomic fragment showing a strain-specific distribution in Southern hybridisations was obtained by differential restriction, hybridisation and cloning of genomic DNA. Each of these clones was sequenced and primers to amplify the insert were designed. When DNA from the strain of origin was used as template, PCR amplification for each of these fragments yielded a single band on gel analysis. One pair of primers amplified the species-constant fragment of 281 bp from DNA of all B. solanacearum strains investigated, from DNA of the closely related bacterium which causes ''blood disease'' of banana (BDB) and in P. syzigii. The sensitivity of detection of B. solanacearum using these ubiquitous primers was between 1.3 and 20 bacterial cells. The feasibility and reliability of a PCR approach to detection and identification of B. solanacearum was tested in diverse strains of the bacterium in several countries and laboratories.

Photodegradation of irinotecan (CPT-11) in aqueous solutions: Identification of fluorescent products and influence of solution composition

The photodegradation of irinotecan (CPT-11), the semisynthetic derivative of the antitumor alkaloid 20(S)-camptothecin, has been investigated. The drug was exposed to laboratory light for up to 5 days in 0.9% saline solution (pH 8.5). Five significant photodegradation products were observed and a high-performance liquid chromatography (HPLC) assay was employed to isolate them from CPT-11 using gradient conditions. The structures were elucidated by nuclear magnetic resonance spectroscopy and tandem mass spectrometry and shown to be the result of extensive modifications of the lactone ring of CPT-11. Three of the compounds were found to belong to the mappicine group of alkaloids. In addition, the effect of light on the stability of CPT-11 in aqueous solutions and biological fluids was also assessed, Potassium phosphate buffers (0.05 M, pH 5.0-8.2) and saline, plasma, urine, and bile solutions containing 20 mu M CPT-11 were equilibrated in the dark for 24 h before being exposed to laboratory light for up to 171 h at ambient temperature. Four of the five identified photodegradation products were observed and quantitated by isocratic HPLC, using a different detection mode (fluorescence) than the one used for gradient elution, In general, CPT-11 was found to be unstable under neutral and alkaline conditions for all solutions investigated, with the exception of bile. We conclude that CPT-11 is photolabile and that care should be taken to protect samples, particularly those intended for the isolation and identification of novel metabolites of CPT-11.

Trisomy 16q21 -> qter: Seven-Year Follow-Up of a Girl With Unusually Long Survival

The 16q21 -> qter duplication is a chromosomal abnormality rarely found in liveborn infants, with only four published cases. We report here on the 7-year follow-up of a female patient with trisomy 16q21 -> qter due to a maternal balanced translocation t(4;16)(q35.2;q21). The patient shows severe mental retardation, congenital heart malformations, nephropathy, and other congenital anomalies. The derivative chromosome was characterized by GTG banding, fluorescent in situ hybridization (FISH) with different BAG probes and the array technique, in order to map the breakpoints. The patient has a 16q21 -> qter duplication, with a 4q35 -> qter monosomy, which we assume does not contribute to the abnormal phenotype. This is the first reported case of postnatal survival to the age of 7 years, an unusually long time in this chromosomal syndrome. (C) 2010 Wiley-Liss, Inc.

Comparative Study of the Standard Fluorescent Antibody to Membrane Antigen (FAMA) Assay and a Flow Cytometry-Adapted FAMA Assay To Assess Immunity to Varicella-Zoster Virus

A flow cytometry-adapted fluorescent antibody to membrane antigen (FAMA) assay to detect IgG antibodies against varicella-zoster virus (VZV) was developed and tested in 62 serum samples, showing 90.32% accuracy obtained from a receiver operating characteristic (ROC) curve with a 0.9125 (95% confidence interval [CI], 0.829 to 1.00) area below the curve compared to the result with standard FAMA.

Childhood radiation-associated atypical meningioma with novel complex rearrangements involving chromosomes 1 and 12

Meningiomas are recognized as the most common late complication following radiotherapy. However, cytogenetic studies in childhood atypical radiation-induced meningioma are sporadic, mainly because this condition generally occurs after a long latent period. In the present study we show the results of conventional and molecular cytogenetics in a 14-year-old boy with a secondary atypical meningioma. Apart from numerical changes, we found complex aberrations with the participation of chromosomes 1, 6 and 12. The invariable presence of loss of 1p was demonstrated by fluorescent in situ hybridization (FISH) analysis with probes directed to telomeric regions and by comparative genome hybridization (CGH). Previous cytogenetic studies on adult spontaneous and radiation-associated meningiomas showed loss of chromosome 22 as the most frequent change, followed by loss of the short arm of chromosome 1. To the best of our knowledge this is the first report of highly complex chromosome aberrations in the pediatric setting of meningioma.

Validation of non-fluorescent methods to reliably detect acrosomal and plasma membrane integrity of common marmoset (Callithrix jacchus) sperm

Simple, rapid and stable sperm evaluation methods which have been optimized for common marmoset (Callithrix jacchus) are critical for studies involving collection and evaluation of sperm in the field. This is particularly important for new species groups such as Callitrichidae where the sperm have been little studied. Of this family, C jacchus is the best known, and has been chosen as a model species for other members of the genus Callithrix. The fundamental evaluation parameters for sperm of any species are viability and acrosomal status. Semen samples were collected by penile vibratory stimulation. To evaluate sperm plasma membrane integrity, Eosin-Nigrosin was tested here for the common marmoset sperm to be used under field conditions. Further, a non-fluorescent stain for acrosome, the ""Simple"" stain, developed for domestic and wild cats, was tested on common marmoset sperm. This was compared with a fluorescent staining, Fluorescein isothiocyanate-Pisum sativum agglutinin (FITC-PSA), routinely used and validated for common marmoset at the German Primate Centre to evaluate acrosomal integrity. Results obtained with the ""Simple"" stain showed a marked differentiation between sperm with intact and non-intact acrosome both with and without ionophore treatment and closely correlated with results obtained with FITC-PSA. Temperature had no effect on the results with the ""Simple"" stain and the complete processing is simple enough to be carried out under field conditions. These findings indicated that the ""Simple"" stain and Eosin-Nigrosin provide rapid and accurate results for C. jacchus sperm and that those methods can be reliably used as field tools for sperm evaluation for this species. (c) 2008 Elsevier Inc. All rights reserved.

Optical encoding of microbeads for gene screening: alternatives to microarrays

Rapid access to genetic information is central to the revolution presently occurring in the pharmaceutical industry, particularly In relation to novel drug target identification and drug development. Genetic variation, gene expression, gene function and gene structure are just some of the important research areas requiring efficient methods of DNA screening. Here, we highlight state-of-the-art techniques and devices for gene screening that promise cheaper and higher-throughput yields than currently achieved with DNA microarrays. We include an overview of existing and proposed bead-based strategies designed to dramatically increase the number of probes that can be interrogated in one assay. We focus, in particular, on the issue of encoding and/or decoding (bar-coding) large bead-based libraries for HTS.

Multi-fluorescent silica colloids for encoding large combinatorial libraries

Large chemical libraries can be synthesized on solid-support beads by the combinatorial split-and-mix method. A major challenge associated with this type of library synthesis is distinguishing between the beads and their attached compounds. A new method of encoding these solid-support beads, 'colloidal bar-coding', involves attaching fluorescent silica colloids ('reporters') to the beads as they pass through the compound synthesis, thereby creating a fluorescent bar code on each bead. In order to obtain sufficient reporter varieties to bar code extremely large libraries, many of the reporters must contain multiple fluorescent dyes. We describe here the synthesis and spectroscopic analysis of various mono- and multi-fluorescent silica particles for this purpose. It was found that by increasing the amount of a single dye introduced into the particle reaction mixture, mono- fluorescent silica particles of increasing intensities could be prepared. This increase was highly reproducible and was observed for six different fluorescent dyes. Multi-fluorescent silica particles containing up to six fluorescent dyes were also prepared. The resultant emission intensity of each dye in the multi-fluorescent particles was found to be dependent upon a number of factors; the hydrolysis rate of each silane-dye conjugate, the magnitude of the inherent emission intensity of each dye within the silica matrix, and energy transfer effects between dyes. We show that by varying the relative concentration of each silane-dye conjugate in the synthesis of multi-fluorescent particles, it is possible to change and optimize the resultant emission intensity of each dye to enable viewing in a fluorescence detection instrument.

Detection of human respiratory syncytial virus in respiratory samples by LightCycler reverse transcriptase PCR

Laboratory diagnosis of human respiratory syncytial virus (hRSV) infections has traditionally been performed by virus isolation in cell culture and the direct fluorescent-antibody assay (DFA). Reverse transcriptase PCR (RT-PCR) is now recognized as a sensitive and specific alternative for detection of hRSV in respiratory samples. Using the LightCycler instrument, we developed a rapid RT-PCR assay for the detection of hRSV (the LC-RT-PCR) with a pair of hybridization probes that target the hRSV L gene. In the present study, 190 nasopharyngeal aspirate samples from patients with clinically recognized respiratory tract infections were examined for hRSV. The results were then compared to the results obtained with a testing algorithm that combined DFA and a culture-augmented DFA (CA-DFA) assay developed in our laboratory. hRSV was detected in 77 (41%) specimens by LC-RT-PCR and in 75 (39%) specimens by the combination of DFA and CA-DFA. All specimens that were positive by the DFA and CA-DFA testing algorithm were positive by the LC-RT-PCR. The presence of hRSV RNA in the two additional LC-RT-PCR-positive specimens was confirmed by a conventional RT-PCR method that targets the hRSV N gene. The sensitivity of LC-RT-PCR was 50 PFU/ml; and this, together with its high specificity and rapid turnaround time, makes the LC-RT-PCR suitable for the detection of hRSV in clinical specimens.

An electromagnetic-field method modeling of a radial line planar antenna with coupling probes

This communications describes an electromagnetic model of a radial line planar antenna consisting of a radial guide with one central probe and many peripheral probes arranged in concentric circles feeding an array of antenna elements such as patches or wire curls. The model takes into account interactions between the coupling probes while assuming isolation of radiating elements. Based on this model, computer programs are developed to determine equivalent circuit parameters of the feed network and the radiation pattern of the radial line planar antenna. Comparisons are made between the present model and the two-probe model developed earlier by other researchers.

Impacts of structural characteristics on activated sludge floc stability

Activated sludge samples from seven full-scale plants were investigated in order to determine the relationship between floc structure and floc stability. Floc stability was determined by shear sensitivity and floc strength. Floc structure was considered in terms of two size scales, the micro- and macrostructure. The microstructure refers to the organization of the floc components, such as the individual microorganisms. The macrostructure refers to the overall floc. The floc macrostructure was characterized by filament index, sludge volume index, size, and fractal dimension. It had a significant impact on floc stability. Large and open floes with low fractal dimensions containing large number of filaments were more shear sensitive and had lower floc strength compared to small and dense floes. Fluorescent in situ hybridization analysis indicated that the organization of the bacterial cells might also have an effect on the floc stability. (C) 2003 Elsevier Ltd. All rights reserved.