Targeted resequencing of candidate genes using selector probes.

Targeted genome enrichment is a powerful tool for making use of the massive throughput of novel DNA-sequencing instruments. We herein present a simple and scalable protocol for multiplex amplification of target regions based on the Selector technique. The updated version exhibits improved coverage and compatibility with next-generation-sequencing (NGS) library-construction procedures for shotgun sequencing with NGS platforms. To demonstrate the performance of the technique, all 501 exons from 28 genes frequently involved in cancer were enriched for and sequenced in specimens derived from cell lines and tumor biopsies. DNA from both fresh frozen and formalin-fixed paraffin-embedded biopsies were analyzed and 94% specificity and 98% coverage of the targeted region was achieved. Reproducibility between replicates was high (R(2) = 0, 98) and readily enabled detection of copy-number variations. The procedure can be carried out in <24 h and does not require any dedicated instrumentation.

Inversion of in situ synthesized oligonucleotides: improved reagents for hybridization and primer extension in DNA microarrays.

Oligonucleotides synthesized in array format suffer from contamination by truncated species. We have developed a method to invert DNA molecules in situ after completed synthesis. Reactive functions at the 5'-ends of the oligonucleotides are permitted to react with functions on the support before the 3'-ends are released, in effect reversing the orientation of full-length oligonucleotides, while any 5'-truncated molecules are lost. This strategy serves both to purify in situ synthesized reagents and to reorient the oligonucleotides, causing them to expose free 3'-hydroxyls. In situ inverted oligonucleotides can be used in assays based on DNA polymerase-assisted extension of immobilized primers, and we demonstrate their utility in minisequencing and in pyrosequencing.

The chemotactic response of Vibrio anguillarum to fish intestinal mucus is mediated by a combination of multiple mucus components.

Chemotactic motility has previously been shown to be essential for the virulence of Vibrio anguillarum in waterborne infections of fish. To investigate the mechanisms by which chemotaxis may function during infection, mucus was isolated from the intestinal and skin epithelial surfaces of rainbow trout. Chemotaxis assays revealed that V. anguillarum swims towards both types of mucus, with a higher chemotactic response being observed for intestinal mucus. Work was performed to examine the basis, in terms of mucus composition, of this chemotactic response. Intestinal mucus was analyzed by using chromatographic and mass spectrometric techniques, and the compounds identified were tested in a chemotaxis assay to determine the attractants present. A number of mucus-associated components, in particular, amino acids and carbohydrates, acted as chemoattractants for V. anguillarum. Importantly, only upon combination of these attractants into a single mixture were levels of chemotactic activity similar to those of intestinal mucus generated. A comparative analysis of skin mucus revealed its free amino acid and carbohydrate content to be considerably lower than that of the more chemotactically active intestinal mucus. To study whether host specificity exists in relation to vibrio chemotaxis towards mucus, comparisons with a human Vibrio pathogen were made. A cheR mutant of a Vibrio cholerae El Tor strain was constructed, and it was found that V. cholerae and V. anguillarum exhibit a chemotactic response to mucus from several animal sources in addition to that from the human jejunum and fish epithelium, respectively.

Toxicokinetics of soman in cerebrospinal fluid and blood of anaesthetized pigs.

The toxicokinetics of the four stereoisomers of the nerve agent C(+/-)P(+/-)-soman was analysed in cerebrospinal fluid (CSF) and blood in anaesthetized, spontaneously breathing pigs during a 90-min period after injection of soman. The pigs were challenged with different intravenous (i.v.) doses of C(+/-)P(+/-)-soman corresponding to 0.75-3.0 LD50 (4.5, 9.0 and 18 microg/kg in a bolus injection and 0.45 microg/kg per min as a slow infusion). Artificial ventilatory assistance was given if, after soman intoxication, the respiratory rate decreased below 19 breaths/min. Blood samples were taken from a femoral artery and CSF samples from an intrathecal catheter. The concentrations of the soman isomers were determined by gas chromatography coupled with high resolution mass spectrometry. All four isomers of soman were detected in both blood and CSF samples. The relatively non-toxic C(+/-)P(+) isomers disappeared from the blood stream and CSF within the first minute, whereas the levels of the highly toxic C(+/-)P(-) isomers could be followed for longer, depending on the dose. Concurrently with the soman analyses in blood and CSF, cholinesterase (ChE) activity and cardiopulmonary parameters were measured. C(+/-)P(-) isomers showed approx. 100% bioavailability in CSF when C(+/-)P(+/-)-soman was given i.v. as a bolus injection. In contrast, C(+/-)P(-) isomers displayed only 30% bioavailability in CSF after slow i.v. infusion of soman. The ChE activity in blood decreased below 20% of baseline in all groups of pigs irrespective of the soman dose. The effect of soman intoxication on the respiratory rate, however, seems to be dose-dependent and the reason for ventilatory failure and death. Artificial ventilation resulted in survival of the pigs for the time-period studied.

The effect of the calcium antagonist nimodipine on the detoxification of soman in anaesthetized rabbits.

The effect of nimodipine, a vasoactive calcium antagonist, on the disappearance of soman from blood was studied in anaesthetized rabbits intoxicated with soman (10.8 micrograms kg-1 i.v.). Blood samples from the left heart ventricle and femoral artery were used to investigate soman detoxification. The concentrations of the soman isomers C+P- and C-P- in blood samples were determined by gas chromatography coupled with high-resolution mass spectrometry. During the sampling, 15-300 s after soman injection, the soman concentration in control animals decreased from 50 to 0.029 ng mL-1; in animals pre-treated with nimodipine (10 mg kg-1) it decreased from 15 to 0.033 ng mL-1. In animals pre-treated with nimodipine the soman concentration was significantly reduced during the first minute of sampling. No differences were detected between soman concentrations in samples from the heart and femoral artery. Acetylcholinesterase inhibition was also used as an indicator of soman activity; there was no difference between the activity of this enzyme in different peripheral organs of control and nimodipine-treated animals. Nimodipine reduces the initial concentration of soman in the blood, which might be of significance in the treatment of soman intoxication.

The protective effect of nimodipine, a calcium antagonist, and its influence on soman clearance in the anaesthetized rabbit.

The effect of pretreatment with a vasoactive compound, nimodipine, on soman intoxication in peripheral organs of rabbits was studied by measuring changes in the cholinesterase and acetylcholinesterase activity and by measuring clearance of soman in blood using gas chromatography/high resolution mass spectrometry. In animals receiving soman only, initial blood concentrations were approximately 100 ng mL-1 and were still detectable after 5 min. The clearance rate of soman in blood markedly increased following nimodipine pretreatment such that soman was below the detection limit (0.002-0.003 ng mL-1) in all samples. Soman injection caused a significant inhibition of the acetylcholinesterase activity in serum, and in brain. In rabbits pretreated with nimodipine, no significant inhibition of acetylcholinesterase activity occurred after soman injection. In view of the effects of nimodipine on soman clearance and on the acetylcholinesterase and cholinesterase inhibition during soman intoxication, we suggest that nimodipine has profound circulatory effects, which during soman intoxication, increase the vascular perfusion through the body and thereby increase the detoxifying capacity.

Imaging human erythrocyte spectrin with atomic force microscopy.

Isolated spectrin covalently attached to a surface in a liquid environment as well as dried on mica has been studied with a contact-mode atomic force microscope. Both pyramidal and conical-type cantilever tip facets were used in the AFM. Our images show structures and give dimensions that correlate well with previous structural studies using transmission electron microscopy.

Optic neuritis and multiple sclerosis: anti-MBP and anti-MBP peptide antibody-secreting cells are accumulated in CSF.

Monosymptomatic unilateral optic neuritis (ON) is a common first manifestation of multiple sclerosis (MS) in which increased numbers of autoimmune T and B cells, recognizing different myelin autoantigens including myelin basic protein (MBP) and its peptides, have been implicated in a hypothetical immunopathogenesis. Using an immunospot assay to detect specific antibodies secreted by individual cells, we analyzed the B-cell repertoire to MBP and its amino acid residues 1-20, 63-88, 110-128, and 148-165 in blood and CSF from patients with ON and MS, and from controls. There were cells secreting IgG antibodies to MBP and the four peptides in blood at mean numbers of 0.9 to 4.6 per 10(5) mononuclear cells, without differences between the three patient groups. Mostly, more than 100-fold more B cells with these specificites per 10(5) cells were found in CSF from the patients with ON and MS, without differences between these two groups but with many fewer in CSF from controls. None of the four included MBP peptides represented an immunodominant B-cell epitope in either ON or MS, and the B-cell response was not more restricted in ON than in MS. The autonomy of the autoimmune B-cell response in CSF was further supported by the pronounced asynchrony of the repertoire to the four MBP peptides in CSF compared with blood in individual patients. The large numbers of MBP- and MBP peptide-reactive B cells in CSF in early MS, as manifested by ON, could play a major role in the immunopathogenesis and perpetuation of MS. Alternatively, they could represent myelin breakdown or restoration.

Leukocyte types in cerebrospinal fluid and peripheral blood enumerated immunoenzymatically in aseptic meningitis and the Guillain-Barré syndrome.

To study celltype distribution simultaneously in peripheral blood (PB) and cerebrospinal fluid (CSF) from patients with aseptic meningitis (AM) (n = 14) and Guillain-Barré syndrome (GBS) (n = 9) we used an immunoenzymatic method that enabled the use of several monoclonal antibodies, also in CSF samples with normal cellcounts. In both patient groups a different cell-distribution in CSF compared to PB was found with regard to pan T cells (CD5+/anti-Leu1+), T cell subpopulations (CD4+/anti-Leu3+, CD8+/anti-Leu2+), B cells (OKB2+, OKB7+), monocytes/macrophages (CD11+/OKM1+) and HLA/DR expressing cells, whereas the distribution of HLA/DC+ cells was similar in CSF and PB. Thus, the CSF cell distribution does not reflect the distribution in PB. The proportion of T cells was higher and the proportion of B-cells was lower in CSF than in PB in both patient groups, which is a finding similar to that in patients with multiple sclerosis. The OKT9 marker, labelling proliferating cells expressing the transferrin receptor, was not useful as marker of local proliferation.

Emission of hydrogen sulfide from sulfur dioxide-fumigated pine trees.

Pine (Pinus silvestris L.) trees subjected to relatively low concentration of SO(2) in the field emit H(2)S from the needles, as demonstrated by gas chromatographic analysis after preconcentration on a molecular sieve. H(2)S is the only reduced sulfurous compound emitted from SO(2) fumigated leaves. The emission is light and SO(2) concentration dependent. Pine trees in the field and in laboratory experiments continue to emit H(2)S several hours after the termination of prolonged SO(2) fumigation. The maximum emission rates observed from pine trees in the field and in laboratory experiments, 14 and 20 nanomoles per milligram chlorophyll per hour respectively, are about the activity expected for the sulfur assimilation pathway in the chloroplasts.

Isoelectric focusing of the phosphoprotein of rat-incisor dentin in ampholine and acid pH gradients. Evidence for carrier ampholyte-protein complexes.

Rat-incisor phosphoprotein (RIP) has been subjected to isoelectric focusing at 4 degrees in (a) an Ampholine pH gradient of 2.5-4 and (b) an acid pH gradient created by electrolysis of a system of acids and acidic ampholytes and covering the pH range 0.5-3.5. In the Ampholine gradient, the RIP unexpectedly formed several adjacent and strongly opalescent bands in the pH range 2.5-3.1. These bands, which migrated slowly toward the anode on prolonged focusing, are interpreted as being the result of an interaction between the amino groups of the Ampholine and the numerous phosphate groups of the protein. In the acid pH gradient, the RIP focused into one narrow zone corresponding to an isoelectric pH of 1.1 at 4 degrees. This value is consistent with the amino-acid composition and the phosphate content of the protein,

Trace analysis for chlorinated hydrocarbons in air by quantitative combustion and coulometric chloride determination: application to standardization of vinyl chloride permeation tubes.

Methods for high-temperature combustion of vinyl chloride in air were studied theoretically and two types of gas mixtures were found to give 100% conversion into HCl. The chloride was determined by coulometric titration with silver, in 70% acetic acid. Good agreement between theoretical and experimental results was obtained. Permeation rates of vinyl chloride from fluorinated ethylene propylene permeation tubes were determined gravimetrically and with the coulometric method developed. The standard deviations of the methods were 0.002 and 0.001 microg min respectively for permeation rates of 0.5 microg min when the temperature was controlled to +/- 0.02 degrees . The coulometric mean value was 99.9% of the gravimetric mean; 1 ppm of vinyl chloride in air could be determined coulometrically with a standard deviation of about 0.002 ppm.

Scanning isoelectric focusing in small density-gradient columns. IV. The use of deuterium oxide for preparing the density gradient and its effects on isoelectric points of proteins.

The use of deuterium oxide as a substitute for sucrose in preparing density gradients for small isoelectrofocusing columns has been investigated. A density gradient was created directly in a 1.5-ml column by free inter-diffusion of three deuterium oxide solutions for 3 min. The resulting deuterium oxide concentration course (as shown by measurement of the refractive-index gradient) had a very high degree of linearity. Test runs with beta-lactoglobulin and sperm-whale myoglobin showed that the strength and stability of the deuterium oxide density gradient should normally be sufficient for stabilization of protein zones against convection during isoelectric focusing. The isoelectric points of beta-lactoglobulins A and B were found to increase as the concentration ratio of deuterium oxide to water at focusing level increased. Within the limits of experimental error, the pI shift corresponding to a given change in solvent composition was equal for both components. These findings are accounted for in terms of the deuterium isotope effect on the dissociation constants of protolytic groups and the shift in the asymmetry potential of the glass electrode in deuterium oxide as compared with water. The spontaneous reduction of sperm-whale metmyoglobin to ferrous myoglobin, which occurs on prolonged isoelectric focusing, is discussed, as are the benefits and drawbacks of deuterium oxide as a density-gradient solute.