Intelligent software for spike separation in multiunit recordings

An algorithm allowing unsupervised spike separation on the basis of three parameters is described. It rests on the assumption that the first incoming spike is part of the first family of spikes. The second spike is compared to the first and, if similar, is included in the first family; otherwise, it constitutes the first member of the second family, and so on. From the first to the 30th spikes of a given family, similarity is established on a percentage basis (10 per 100 around the family's centre of gravity), and when the family includes over 30 spikes, similarity is measured by the standard deviation (1·96 SD around the family's centre of gravity). As new families occur, the families are sorted according to the number of spikes they comprise. The algorithm, its implementation and related software are fully described. Results were tested with both artificial and natural material. Using artificial spikes as input, it was demonstrated that on average 68 per cent of spikes were correctly classified, 30 per cent were rejected, and only 2 per cent were wrongly classified. For natural spike inputs, 65 per cent of recorded spikes were classified, and their separation into several families were confirmed on a physiological basis.     

A pedigree-based algorithm for finding efficient peeling sequences

The computational cost, in terms of both storage requirementsand calculation required, of performing an elimination orderingon a graph is considered as a function of the order in whichthe vertices of the moral graph are eliminated. Useful propertiesof the moral graph of a pedigree with respect to vertex eliminationare observed and these properties extended to define a k-pedigreeas a graph permitting allocation of one of k sexes to each vertexof the graph, such that the subgraph induced by vertices ofa single six contains no cycles. Properties of k-pedigrees includean upper bound of 2k on clique size. A novel algorithm, SEXY,based upon these properties is proposed and its performancecompared with other algorithms used to generate eliminationsequences. It is found to give a widely dispersed range of sequences,including some sequences requiring under a quarter of the storageand under a half of the computational time than had previouslybeen found using standard methods.     

Oxidation of PAH and PAH-derivatives by fungal and plant oxidoreductases

The in vitro-oxidation of the three- and four-ring polycyclic aromatic hydrocarbons (PAH) anthracene, phenanthrene, pyrene and fluoranthene by preparations of extracellular lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase (Lacc) of the white rot fungus Nematoloma frowardii and by mushroom tyrosinase (Tyr) and horseradish peroxidase (HRP) was investigated. LiP transformed 58.6% of anthracene and 34.2% of pyrene, whereas 31.5% of anthracene and 11.2% pyrene were oxidized by MnP. In the presence of the mediating substances veratryl alcohol (for LiP), GSH (for MnP), and ABTS (for Lacc, Tyr, HRP), the conversion of PAH was enhanced in most cases. Inclusion of PAH-derivatives, known as intermediates or potential dead-end-products of microbial PAH metabolism, in the in vitro-oxidation studies, demonstrated that the hydroxylated PAH metabolites served as substrates for all oxidoreductases tested, whereas PAH-quinones and oxo-metabolites were not transformed.     

A non-phenol-chloroform extraction of double-stranded RNA from plant and fungal tissues

Double-stranded RNA (dsRNA) molecules of viruses are found in nature at a very high frequency. Their detection in plants and fungi has been carried out with difficulty due to the complicated dsRNA extraction techniques used commonly which includes phenol-chloroform extractions. In this study, an extraction method for isolation of dsRNA is described that is free of phenol and chloroform. A lysis buffer, containing beta-mercaptoethanol and polyvinylpolypyrrolidone (PVPP-40), was added to homogenised tissues and the subsequent supernatant was filtered through a cellulose CF-11 mini-column. DsRNA molecules were separated based on the differing affinity of nucleic acids for the cellulose CF-11 resin in 20% ethanol buffer. This easy, rapid and cheap technique has been successfully tested on fungi and plants containing different dsRNA virus molecules, indicating the possibility of a wide use of the method.     

Letter to the Editor: Mastrevirus sequences in a begomovirus-infected plant

Recombination is a major driver of diversification of geminiviruses and is believed to be, for a large part, responsible for the present taxonomic structure of the family Geminiviridae. Examples of recent intergeneric recombination between viruses of the genera Begomovirus and Mastrevirus remain to be identified. Here, we show that one of the prerequisites for begomovirus–mastrevirus intergeneric recombination, co-infection of a single plant, does occur. The lack of reported recombination between viruses of these two genera may suggest that there are constraints to viable intergeneric recombinant viruses being produced, possibly due to the extreme genetic distances between extant begomo- and mastreviruses.     

A new MLC segmentation algorithm/software for step-and-shoot IMRT delivery

We present a new MLC segmentation algorithm/software for step-and-shoot IMRT delivery. Our aim in this work is to shorten the treatment time by minimizing the number of segments. Our new segmentation algorithm, called SLS (an abbreviation for static leaf sequencing), is based on graph algorithmic techniques in computer science. It takes advantage of the geometry of intensity maps. In our SLS approach, intensity maps are viewed as three-dimensional (3-D) "mountains" made of unit-sized "cubes." Such a 3-D "mountain" is first partitioned into special-structured submountains using a new mixed partitioning scheme. Then the optimal leaf sequences for each submountain are computed by either a shortest-path algorithm or a maximum-flow algorithm based on graph models. The computations of SLS take only a few minutes. Our comparison studies of SLS with CORVUS (both the 4.0 and 5.0 versions) and with the Xia and Verhey segmentation methods on Elekta Linac systems showed substantial improvements. For instance, for a pancreatic case, SLS used only one-fifth of the number of segments required by CORVUS 4.0 to create the same intensity maps, and the SLS sequences took only 25 min to deliver on an Elekta SL 20 Linac system in contrast to the 72 min for the CORVUS 4.0 sequences (a three-fold improvement). To verify the accuracy of our new leaf sequences, we conducted film and ion-chamber measurements on phantom. The results showed that both the intensity distributions as well as dose distributions of the SLS delivery match well with those of CORVUS delivery. SLS can also be extended to other types of Linac systems.     

Analysis of genes coding for small-subunit rRNA sequences in studying phylogenetics of dematiaceous fungal pathogens.

Because of their ability to display yeast-like growth forms in various environmental conditions, dematiaceous (melanized) hyphomycetes of the form-genera Exophiala, Rhinocladiella, and Wangiella have been informally termed "black yeasts." Cladistic analysis of 1,050 bp of the genes coding for small-subunit rRNA (SSU rDNA) supported a close relationship among species of these black yeasts with other dematiaceous hyphomycetes in the form-genera Fonsecaea, Phialophora, and Ramichloridium. The conventional categories of these fungi based on asexual states are not supported by phylogenetic analysis of SSU rDNA sequences. Isolates exhibiting annellidic modes of blastic conidiogenesis (e.g., Exophiala spp.) were not monophyletic and were placed as sister taxa to isolates that produce phialides or sympodulae. The results indicated very close relationships between isolates of Wangiella dermatitidis and Exophiala mansonii and between Rhinocladiella aquaspersa and Exophiala jeanselmei. This clade of dematiaceous hyphomycetes was a sister group to a clade comprising members of two orders of cleistothecial ascomycetes, Eurotiales and Onygenales. The etiological agents of chromoblastomycosis were found to be a closely related group (clade), while the agents of phaeohyphomycosis displayed a broader distribution on the SSU rDNA tree.     

An algorithm for maximizing the probability of complication-free tumour control in radiation therapy.

New radiobiological models are used to describe tumour and normal tissue reactions and to account for their dependence on the irradiated volume and inhomogeneities of the delivered dose distribution and cell sensitivity. The probability of accomplishing complication-free tumour control is maximized by an iterative algorithm. The algorithm is demonstrated by applying it to a one-dimensional (1D) tumour model but also to a more clinically relevant 2D case. The new algorithm is n-dimensional so it could simultaneously optimize the dose delivery in a 3D volume and in principle also select the ideal beam orientations, beam modalities (photons, electrons, neutrons, etc) and optimal spectral distributions of the corresponding modalities. To make calculation time reasonable, 2D-3D problems are most practical, and suitable beam orientations are preselected by the choice of irradiation kernel. The energy deposition kernel should therefore be selected in order to avoid irradiation through organs at risk. Clinically established dose response parameters for the tissues of interest are used to make the optimization as relevant as possible to the clinical problems at hand. The algorithm can be used even with a poorly selected kernel because it will always, as far as possible, avoid irradiating organs at risk. The generated dose distribution will be optimal with respect to the spatial distribution and assumed radiobiological properties of the tumour and normal tissues at risk for the kernel chosen. More specifically the probability of achieving tumour control without fatal complications in normal tissues is maximized. In the clinical examples a reduced tumour dose is seen at the border to sensitive organs at risk, but instead an increased dose just inside the tumour border is generated. The increased tumour dose has the effect that the dose fall-off is as steep as possible at the border to organs at risk.     

An improved MLC segmentation algorithm and software for step-and-shoot IMRT delivery without tongue-and-groove error

We present an improved multileaf collimator (MLC) segmentation algorithm, denoted by SLS(NOTG) (static leaf sequencing with no tongue-and-groove error), for step-and-shoot intensity-modulated radiation therapy (IMRT) delivery. SLS(NOTG) is an improvement over the MLC segmentation algorithm called SLS that was developed by Luan et al. [Med. Phys. 31(4), 695-707 (2004)], which did not consider tongue-and-groove error corrections. The aims of SLS(NOTG) are (1) shortening the treatment times of IMRT plans by minimizing their numbers of segments and (2) minimizing the tongue-and-groove errors of the computed IMRT plans. The input to SLS(NOTG) is intensity maps (IMs) produced by current planning systems, and its output is (modified) optimized leaf sequences without tongue-and-groove error. Like the previous SLS algorithm [Luan et al., Med. Phys. 31(4), 695-707 (2004)], SLS(NOTG) is also based on graph algorithmic techniques in computer science. It models the MLC segmentation problem as a weighted minimum-cost path problem, where the weight of the path is the number of segments and the cost of the path is the amount of tongue-and-groove error. Our comparisons of SLS(NOTG) with CORVUS indicated that for the same intensity maps, the numbers of segments computed by SLS(NOTG) are up to 50% less than those by CORVUS 5.0 on the Elekta LINAC system. Our clinical verifications have shown that the dose distributions of the SLS(NOTG) plans do not have tongue-and-groove error and match those of the corresponding CORVUS plans, thus confirming the correctness of SLS(NOTG). Comparing with existing segmentation methods, SLS(NOTG) also has two additional advantages: (1) SLS(NOTG) can compute leaf sequences whose tongue-and-groove error is minimized subject to a constraint on the maximum allowed number of segments, which may be desirable in clinical situations where a treatment with the complete correction of tongue-and-groove error takes too much time, and (2) SLS(NOTG) can be used to minimize a more general type of error called the tongue-or-groove error.     

Localisation of DNA sequences on plant chromosomes using PRINS and C-PRINS

Localisation of DNA sequences to plant chromosomes in situ has traditionally been accomplished using fluorescence in situ hybridisation (FISH). Although the method is suitable for most applications it is time-consuming and requires labelled probes. Recently, primed in situ labelling (PRINS) has been developed as an alternative to FISH. PRINS is based on annealing of unlabelled oligonucleotide primer(s) to chromosome DNA and its elongation by DNA polymerase in the presence of labelled nucleotide(s). The method was found useful to detect high-copy tandem repeats on plant chromosomes. Low copy repeats were detected after a more sensitive variant of PRINS called cycling PRINS (C-PRINS), which involves a sequence of thermal cycles analogous to polymerase chain reaction. This paper describes protocols of PRINS and C-PRINS, which have been optimised for chromosome spreads and for chromosomes purified using gradient centrifugation and/or flow sorting. The methods result in clear signals with negligible non-specific labelling. Further work is needed to improve the sensitivity to allow for reliable detection of single-copy DNA sequences.     

Detection of polyadenylic acid sequences in plant pathogenic RNAs

Polyadenylic acid sequences were detected in bean pod mottle (BPMV) and cowpea mosaic virus (CPMV) RNAs but not in tobacco rattle (TRV) and tobacco mosaic virus (TMV) RNAs or the low molecular weight pathogenic RNA (viroid) from citrus exocortis disease (CEV). A resistant RNA fragment from BPMV-RNA-treated with T₁ and pancreatic ribonuclease migrated as 4–5 S RNA in polyacrylamide gel electrophoresis. The polyadenylic acid sequence appears to be associated with both components of the BPMV genome.     

An active contour model algorithm for tracking endocardiac boundaries in echocardiographic sequences

The use of active contour models to track the boundaries of anatomic structures in medical images is a technique that has attracted a great number of efforts during the last decade. Segmentation techniques based in deformable active contours were proposed first by Kass et al. Because of the problems appearing using these models, some solutions have been introduced, such as balloon force or Gradient Vector Flow force (GVF), derived from the Gradient Vector Flow vectorial field. Results obtained with these forces in the tracking endocardiac task in echocardiographic sequences were not adequate. We have designed a new external force called hybrid force, which, by combining both forces, joins the main features of each one.     

Genetic transformation of the fungal plant wilt pathogen,Fusarium oxysporum

Summary A system for transformation of the fungal plant pathogen Fusarium oxysporum has been developed. The system employs plasmids which contain a bacterial hygromycin B phosphotransferase gene (hph) linked to Aspergillus regulatory sequences and which confer hygromycin B resistance in Fusarium. Transformation resulted from integration of the vectors into heterologous regions of the Fusarium genome and occurred at a frequency of approximately one transformant per µg DNA. No evidence was found for autonomous replication of the vector in the fungus. The transformed, drug resistant phenotype was mitotically stable with or without selection. However, modification of integrated DNA could occur during vegetative growth.     

A genetic similarity algorithm for searching the Gene Ontology terms and annotating anonymous protein sequences

A genetic similarity algorithm is introduced in this study to find a group of semantically similar Gene Ontology terms. The genetic similarity algorithm combines semantic similarity measure algorithm with parallel genetic algorithm. The semantic similarity measure algorithm is used to compute the similitude strength between the Gene Ontology terms. Then, the parallel genetic algorithm is employed to perform batch retrieval and to accelerate the search in large search space of the Gene Ontology graph. The genetic similarity algorithm is implemented in the Gene Ontology browser named basic UTMGO to overcome the weaknesses of the existing Gene Ontology browsers which use a conventional approach based on keyword matching. To show the applicability of the basic UTMGO, we extend its structure to develop a Gene Ontology -based protein sequence annotation tool named extended UTMGO. The objective of developing the extended UTMGO is to provide a simple and practical tool that is capable of producing better results and requires a reasonable amount of running time with low computing cost specifically for offline usage. The computational results and comparison with other related tools are presented to show the effectiveness of the proposed algorithm and tools.     

Sequence of centromere separation: a mechanism for orderly separation of dicentrics

Stable dicentric chromosomes from three mouse cell lines (viz., SEWA Rec4, brain tumor, and L-cells), as well as a human t(9;11) line were analyzed for the sequence in which the two centromeres separate. At prometaphase, as well as in many cells at midmetaphase, the dicentrics express the two centromeres in the form of two primary constrictions. As the cell advances to late metaphase, one of the constrictions loosens the two chromatids so that eventually there is no connection between them. The other centromere stays intact during this period and separates into two units at the metaanaphase junction along with the rest of the genome. The centromere that separates prematurely (out-of-phase) usually is the same in a given dicentric. It is proposed that such a prematurely separating centromere does not function as active element during chromatid migration. Apparently, in dicentrics some sort of control is exerted to eliminate the functioning of one centromere. The nature of such control is not understood at this time. The mouse dicentrics "synthesize" only one kinetochore as definable by antikinetochore antibody studies.     

Transient and multivariate system for transformation of a fungal plant pathogen, Rosellinia necatrix, using autonomously replicating vectors

Rosellinia necatrix is a fungus that infects a wide range of host plants and ruins a variety of commercially important crops. DNA fragments can be introduced into R. necatrix using conventional protoplast-PEG transformation and genome-integrating vectors; however, transformation efficiency with this strategy is quite low. Therefore, to establish a more effective transformation system for the studies of R. necatrix, an autonomously replicating vector was constructed using AMA1 sequences derived from Aspergillus nidulans, which is distantly related to R. necatrix. Use of this vector with AMA1 sequences increased transformation efficiency in R. necatrix, and the vector was maintained as a plasmid in the transformants. Transient and multivariate functional analyses in R. necatrix were performed using co-transformation of multiple pAMA-H vectors, which each carried either an expression cassette for eGFP, mOrange2, or a geneticin resistance gene. Furthermore, fluorescent proteins expressed from the autonomously replicating vectors were dispersed throughout fungal colonies even though the vectors themselves were restricted to the center of each colony. This intriguing phenomenon indicated that gene products could move from the center to the margin in a colony of the filamentous fungi via a cell-to-cell transport system.