An assay for dielectrophoresis: Applications to electromagnetically induced membrane adhesion and fusion

A new assay for dielectrophoresis, consisting of a platinum wire placed along the axis of symmetry of a hollow metal cylinder, was constructed. The main advantage of this device is that because of the simple axisymmetrical electric field the experimental data can be easily interpreted. Preliminary experimental data for adhesion and fusion of red blood cells are reported. The most interesting observation was that fusion of cell membranes can be induced without applying DC electrical pulses, merely because of the dielectrophoretic effect. A possible explanation of this phenomenon is suggested.     

The chromatin fiber: structure and conformational transitions as revealed by optical anisotropy studies

Structure and conformational transitions of the chromatin fiber as revealed by optical anisotropy studies are reviewed. The data in the literature do not allow a definite interpretation; in fact some of them are contradictory. The major findings are reported here and an attempt is made to analyse them with respect to the internal dynamics and the various models suggested for the organization of the chromatin fiber.     

Production of male sterile interspecific somatic hybrids between transgenic N. tabacum (Bar) and N. rotundifolia (NPT II) and their identification by aflp analysis

Summary A protoplast fusion experiment was carried out aiming to obtain somatic hybrid plants of transgenic Nicotiana tabacum (bar) (+) N. rotundifolia (npt II). The bialaphos resistance marker (bar) was introduced into N. tabacum via Agrobacterium tumefaciens using vector pGV1500 carrying the bar gene phosphinothricin acetyltransferase. N. rotundifolia (npt II) was recovered after direct gene transformation of protoplasts by the pGP6 plasmid carrying the npt II gene for neomycin phosphotransferase. Both plasmids possessed 35S CaMV promotors. Hybrid selection was based on dual bialaphos— kanamycin resistance. Amplified fragment length polymorphism (AFLP) analysis of regenerated plants showed the presence of species-specific bands for both parents, which confirmed their hybrid nature. N. tabacum (bar) (+) N. rotundifolia (npt II) hybrids exhibited a great diversity in morphology. Fertile hybrids which possessed N. tabacum or N. rotundifolia morphology were recovered. Flow cytometric analysis revealed that the N. tabacum- and N. rotundifolio-like hybrids had nuclear DNA contents near that of N. tabacum (9.40±0.24pg) or N. rotundifolia (5.29±0.36 pg), respectively, and were highly asymmetric. Other hybrids combined traits from the two species at various levels—N. tabacum habit or branched, similar to N. rotundifolia. Their leaves varied in shape. The flowers of the hybrid plants were of N. tabacum or N. rotundifolia type, or had N. rotundifolia dimensions, pink with N. tabacum corolla or white with curly fused petals. All were self-sterile or male sterile. The nuclear DNA content varied from 8.90±0.30 to 19.57±0.33 pg. The data from the morphological and cytological analysis provided vidence that parental chromosome elimination in the hybrid clones was spontaneous and not species-specific and that diploidization of the tobacco genome might have occurred in some clones during in vitro culture. This reflects the genomic incompatibility between the two species.     

A glimpse on the pattern of rodent diversication: a phylogenetic approach

ABSTRACT: BACKGROUND: Development of phylogenetic methods that do not rely on fossils for the study of evolutionary processes through time have revolutionized the eld of evolutionary biology and resulted in an unprecedented expansion of our knowledge about the tree of life. These methods have helped to shed light on the macroevolution of many taxonomic groups such as the placentals (Mammalia). However, despite the increase of studies addressing the diversication patterns of organisms, no synthesis has addressed the case of the most diversied mammalian clade: the Rodentia. RESULTS: Here we present a rodent maximum likelihood phylogeny inferred from a molecular supermatrix. It is based on 11 mitochondrial and nuclear genes that covers 1,265 species, i.e., respectively 56 % and 81 % of the known specic and generic rodent diversity. The inferred topology recovered all Rodentia clades proposed by recent molecular works. A relaxed molecular clock dating approach provided a time framework for speciation events. We found that the Myomorpha clade shows a greater degree of variation in diversication rates than Sciuroidea, Caviomorpha, Castorimorpha and Anomaluromorpha. We identied a number of shifts in diversication rates within the major clades: two in Castorimorpha, three in Ctenohystrica, 6 within the squirrel-related clade and 24 in the Myomorpha clade. The majority of these shifts occurred within the most recent familial rodent radiations: the Cricetidae and Muridae clades. Using the topological imbalances and the time line we discuss the potential role of different diversication factors that might have shaped the rodents radiation. CONCLUSIONS: The present glimpse on the diversication pattern of rodents can be used for further comparative meta-analyses. Muroid lineages have a greater degree of variation in their diversication rates than any other 1rodent group. Different topological signatures suggest distinct diversication processes among rodent lineages. In particular, Muroidea and Sciuroidea display widespread distribution and have undergone evolutionary and adaptive radiation on most of the continents. Our results show that rodents experienced shifts in diversication rate regularly through the Tertiary, but at different periods for each clade. A comparison between the rodent fossil record and our results suggest that extinction led to the loss of diversication signal for most of the Paleogene nodes.     

Characterization of Influenza Virus-Induced Death of J774.1 Macrophages

The mechanism and role of influenza virus (IV)-induced pathogenesis of macrophages during respiratory infection are ill defined. Reported here are findings on IV-induced cytopathic effects (CPEs) for anin vitroexperimental system using the murine macrophage cell line J774.1. CPE was elicited by 0.2 or greater multiplicity of infection (m.o.i.). CPEs showed a lag of 6–8 h postinfection and occurred most rapidly between 6 and 12 h. J774.1 cells did not support productive IV replication, but immunofluorescence demonstrated that IV protein synthesis occurred. Light microscopy and DNA staining showed that after death cells had very condensed cytoplasm and nuclei. Cell remnants were surrounded by intact plasma membrane (PM) as demonstrated by exclusion of a membrane-impermeant dye. Time-lapse video microscopy recordings between 6 and 10 h postinfection showed sequential structural changes, including previously undescribed events. Notable changes were a rapid cytokinesis (zeiosis; “cell boiling”), followed by nuclear shrinkage, and an unusual transient blebbing of the PM. DNA fragmentation occurred after 12 h, producing a wide size range. UV-inactivated virus failed to induce CPEs, and CPE was blocked by amantadine.N-Acetylcysteine and pyrrolidine dithiocarbamate, but not other inhibitors of reactive oxygen intermediates, reduced or blocked the CPE. Most changes observed are those attributed to apoptotic processes rather than necrotic cell death. The kinetics and inhibitor effects suggest that IV infection and replication must be initiated to activate CPEs.     

Biodegradation of aniline using light carriers with optimised surface in TPIFB

The successful microbial immobilisation along with fluidisation are keys to highly effective microbiological wastewater treatment. The Inverse Three-Phase Fluidised Bed (TPIFB) is a novel technique proposed for wastewater treatment. In the present work a method based on analysis of the factors governing the biofilm formation has been developed for production of biocarriers for packing in a TPIFB for biodegradation of aniline containing wastewater. Tests of the treated samples show that the procedure ensures forming of a steady biofilm. The continuous cultivation of Pseudomonas putuda and Ps. aeruginosa in TPIFB with aniline as a single substrate provides high biodegradation rates and stability of the process of wastewater treatment within one-year-long operation. Electrochemical probe for monitoring and control the biofilm growth proves its reliability and efficiency in TPIFB.     

Rapid virulence prediction and identification of Newcastle disease virus genotypes using third-generation sequencing

Background

Newcastle disease (ND) outbreaks are global challenges to the poultry industry. Effective management requires rapid identification and virulence prediction of the circulating Newcastle disease viruses (NDV), the causative agent of ND. However, these diagnostics are hindered by the genetic diversity and rapid evolution of NDVs.

Methods

An amplicon sequencing (AmpSeq) workflow for virulence and genotype prediction of NDV samples using a third-generation, real-time DNA sequencing platform is described here. 1D MinION sequencing of barcoded NDV amplicons was performed using 33 egg-grown isolates, (15 NDV genotypes), and 15 clinical swab samples collected from field outbreaks. Assembly-based data analysis was performed in a customized, Galaxy-based AmpSeq workflow. MinION-based results were compared to previously published sequences and to sequences obtained using a previously published Illumina MiSeq workflow.

Results

For all egg-grown isolates, NDV was detected and virulence and genotype were accurately predicted. For clinical samples, NDV was detected in ten of eleven NDV samples. Six of the clinical samples contained two mixed genotypes as determined by MiSeq, of which the MinION method detected both genotypes in four samples. Additionally, testing a dilution series of one NDV isolate resulted in NDV detection in a dilution as low as 10¹ 50% egg infectious dose per milliliter. This was accomplished in as little as 7 min of sequencing time, with a 98.37% sequence identity compared to the expected consensus obtained by MiSeq.

Conclusion

The depth of sequencing, fast sequencing capabilities, accuracy of the consensus sequences, and the low cost of multiplexing allowed for effective virulence prediction and genotype identification of NDVs currently circulating worldwide. The sensitivity of this protocol was preliminary tested using only one genotype. After more extensive evaluation of the sensitivity and specificity, this protocol will likely be applicable to the detection and characterization of NDV.