Mutational pattern of influenza B viruses adapted to high growth replication in embryonated eggs

Improved replication of influenza viruses in embryonated chicken eggs (CE) permits increased vaccine production and availability. We investigated the growth properties of influenza B viruses in relation to specific mutations occurring after serial passage in CE. In serial passage experiments yielding high growth variants of B/Victoria/504/2000, mutations predicted to alter amino acid (AA) composition occurred only near the receptor-binding pocket of the hemagglutinins (HA) and in no other genes. Two B/Victoria/504/2000 high growth variants had the same AA substitutions in HA (R162M and D196Y), but the higher yield variant had a third substitution (G141E), which also altered antigenic characteristics. In a serial passage experiment yielding a high growth variant of B/Hong Kong/330/2001, mutations predicted to alter AA composition occurred only in PB2 and NP in domains predicted to relate to RNP formation and function. Our results indicate that adaptation of influenza B viruses to high-yield replication by serial passage in CE requires few mutations either in internal or external genes. Specific modifications of genes or a combination of genes could be used to optimize or create influenza B viruses for specific growth substrates.     

Temperature compensation of neuromuscular modulation in aplysia

Physiological systems that must operate over a range of temperatures often incorporate temperature-compensatory mechanisms to maintain their output within a relatively narrow, functional range of values. We analyze here an example in the accessory radula closer (ARC) neuromuscular system, a representative part of the feeding neuromusculature of the sea slug Aplysia. The ARC muscle's two motor neurons, B15 and B16, release, in addition to ACh that contracts the muscle, modulatory peptide cotransmitters that, through a complex network of effects in the muscle, shape the ACh-induced contractions. It is believed that this modulation is critical in optimizing the performance of the muscle for successful, efficient feeding behavior. However, previous work has shown that the release of the modulatory peptides from the motor neurons decreases dramatically with increasing temperature. From 15 to 25 degrees C, for example, release decreases 20-fold. Yet Aplysia live and feed successfully not only at 15 degrees C, but at 25 degrees C and probably at higher temperatures. Here, working with reduced B15/B16-ARC preparations in vitro as well as a mathematical model of the system, we have found a resolution of this apparent paradox. Although modulator release decreases 20-fold when the temperature is raised from 15 to 25 degrees C, the observed modulation of contraction shape does not decrease at all. Two mechanisms are responsible. First, further downstream within the modulatory network, the modulatory effects themselves-experimentally dissected by exogenous modulator application-have temperature dependencies opposite to that of modulator release, increasing with temperature. Second, the saturating curvature of the dose-response relations within the network diminishes the downstream impact of the decrease of modulator release. Thus two quite distinct mechanisms, one depending on the characteristics of the individual components of the network and the other emerging from the network's structure, combine to compensate for temperature changes to maintain the output of this physiological system.     

Observation of a nematic phase displayed by a polysiloxane with trinitrofluorenones as side groups

The synthesis and the results of the structural study of two copolysiloxanes with laterally fixed trinitrofluorenone (TNF) units is reported. The two copolysiloxanes having 2,4 ( 1a ) and 5,3 ( 1b ) dimethylsiloxane comonomer units per TNF side group differ significantly in their phase behaviour as evident from optical microscopy, differential scanning calorimetry and X-ray scattering: 1b shows a nematic mesophase whereas 1a is an amorphous material. The different phase behaviour is discussed in terms of microphase separation between the siloxane backbone and TNF side groups.     

Investigating Population Risk Factors of Pancreatic Cancer by Evaluation of Optical Markers in the Duodenal Mucosa

Pancreatic cancer screening has been hampered by the high rate of complications associated with interrogating the pancreas. The closest non-invasively accessible mucosa available for pancreatic cancer screening is the periampullary duodenal tissue. Our earlier report has shown the potential of using optical markers to interrogate this tissue for the presence of pancreatic cancer. In this study, we report a larger data set of low-coherence enhanced backscattering (LEBS) and elastic light scattering fingerprinting (ELF) optical markers from the periampullary duodenal mucosa. Optical measurements from biopsy samples were acquired from a total of 203 patients with varying clinical classification including healthy controls, a family history of pancreatic cancer, pancreatitis, mucinous cystic precursor lesions, pancreatic cancer, and other pancreatic malignancies. Evaluation of the performance of an independent testing set for discriminating healthy control patients from pancreatic cancer patients showed a 95% sensitivity, 71% specificity, and 85% area under the receiver operator characteristic (AUROC) curve. Importantly, this performance was uncompromised for detecting potentially curable stages of the disease. Additionally, optical markers in higher risk populations such as family history and pancreatitis had values between those of healthy control and pancreatic cancer patients, thus allowing for future investigations of screening from these high risk groups.     

Two methods of whole-genome amplification enable accurate genotyping across a 2320-SNP linkage panel

Comprehensive genome scans involving many thousands of SNP assays will require significant amounts of genomic DNA from each sample. We report two successful methods for amplifying whole-genomic DNA prior to SNP analysis, multiple displacement amplification, and OmniPlex technology. We determined the coverage of amplification by analyzing a SNP linkage marker set that contained 2320 SNP markers spread across the genome at an average distance of 2.5 cM. We observed a concordance of >99.8% in genotyping results from genomic DNA and amplified DNA, strongly indicating the ability of both methods used to amplify genomic DNA in a highly representative manner. Furthermore, we were able to achieve a SNP call rate of >98% in both genomic and amplified DNA. The combination of whole-genome amplification and comprehensive SNP linkage analysis offers new opportunities for genetic analysis in clinical trials, disease association studies, and archiving of DNA samples.     

Fc receptor-mediated accumulation of macrophages in crescentic glomerulonephritis induced by anti-glomerular basement membrane antibody administration in WKY rats

Anti-glomerular basement membrane (GBM) glomerulonephritis induced in WKY rats is characterized by glomerular accumulation of CD8(+) T cells and monocytes/macrophages, followed by crescent formation. The mechanism of leukocyte accumulation after antibody binding to GBM is still unclear. To unveil an involvement of Fcgamma receptors (FcgammaR) in leukocytes recruitment we examined the expression of FcgammaR in glomeruli and the effects of the administration of F(ab')(2) fragment of anti-GBM antibody or FcgammaR blocking on the initiation and progression of this model. A gradual increase of FcgammaR mRNA expression in glomeruli during the time course of disease suggested their significance in the development of glomerulonephritis. Glomerular lesions and proteinuria were induced only in rats injected with intact IgG of anti-GBM antibody, but not with the F(ab')(2) fragment. In vivo blocking of FcgammaR by administering heat-aggregated IgG led to the decrease of mRNA expression for all types of FcgammaR (types 1, 2 and 3) and a significant amelioration of glomerulonephritis manifestations. By flow cytometry and immunohistochemistry FcgammaR2-expressing cells in glomeruli were identified as macrophages, but not CD8(+) T cells. The expression of FcgammaR1 and 3 was significantly decreased, and that of FcgammaR2 became undetectable in CD8(+) T cell-depleted rats. Thus, CD8(+) T cells may stimulate FcgammaR expression on macrophages, contributing to their glomerular accumulation and injury. These studies provide direct evidence for a crucial involvement of IgG Fc-FcgammaR interaction in glomerular recruitment of macrophages and following induction of anti-GBM glomerulonephritis in WKY rats.     

Tight or loose coupling between components of the feeding neuromusculature of Aplysia?

Like other complex behaviors, the cyclical, rhythmic consummatory feeding behaviors of Aplysia-biting, swallowing, and rejection of unsuitable food-are produced by a complex neuromuscular system: the animal's buccal mass, with numerous pairs of antagonistic muscles, controlled by the firing of numerous motor neurons, all driven by the motor programs of a central pattern generator (CPG) in the buccal ganglia. In such a complex neuromuscular system, it has always been assumed that the activities of the various components must necessarily be tightly coupled and coordinated if successful functional behavior is to be produced. However, we have recently found that the CPG generates extremely variable motor programs from one cycle to the next, and so very variable motor neuron firing patterns and contractions of individual muscles. Here we show that this variability extends even to higher-level parameters of the operation of the neuromuscular system such as the coordination between entire antagonistic subsystems within the buccal neuromusculature. In motor programs elicited by stimulation of the esophageal nerve, we have studied the relationship between the contractions of the accessory radula closer (ARC) muscle, and the firing patterns of its motor neurons B15 and B16, with those of its antagonist, the radula opener (I7) muscle, and its motor neuron B48. There are two separate B15/B16-ARC subsystems, one on each side of the animal, and these are indeed very tightly coupled. Tight coupling can, therefore, be achieved in this neuromuscular system where required. Yet there is essentially no coupling at all between the contractions of the ARC muscles and those of the antagonistic radula opener muscle. We interpret this result in terms of a hypothesis that ascribes a higher-order benefit to such loose coupling in the neuromusculature. The variability, emerging in the successive feeding movements made by the animal, diversifies the range of movements and thereby implements a trial-and-error search through the space of movements that might be successful, an optimal strategy for the animal in an unknown, rapidly changing feeding environment.     

Generation and characterization of single-chain antibody fragments specific against transmembrane envelope glycoprotein gp46 of maedi-visna virus

A single-chain antibody fragments (scFv) was developed directed against transmembrane envelope glycoprotein gp46 of the virus maedi-visna, by the application of the antibody phage display library. To get specific scFv binders, the library was panned against the biotinylated peptide of 20 amino acids corresponding to the principal immunodominant domain of gp46 protein. The number of positively binding scFvs was evaluated by scFv-phage ELISA, BstN1 fingerprinting and DNA sequencing. The scFvs were expressed in soluble form and purified by immobilized metal affinity chromatography (IMAC) with a yield of 2-2.5 mg/l. Two scFvs have shown to recognize gp46 and gp150 proteins in Western blot analysis. The scFvs also recognized the virus in infected cells as shown by immunofluorescence assay. The affinity of the obtained antibody fragments to gp46 peptide was measured by surface plasmon resonance, and the resulting K(A) was in the 10(6)-10(7)lmol(-1) range. The application of characterized scFvs for expression as intrabodies in intracellular immunization against virus maedi-visna infection and for the diagnosis of this virus is discussed.