SWORDS: A statistical tool for analysing large DNA sequences

In this article, we present some simple yet effective statistical techniques for analysing and comparing large DNA sequences. These techniques are based on frequency distributions of DNA words in a large sequence, and have been packaged into a software called SWORDS. Using sequences available in public domain databases housed in the Internet, we demonstrate how SWORDS can be conveniently used by molecular biologists and geneticists to unmask biologically important features hidden in large sequences and assess their statistical significance.     

Criteria and techniques for analysing cell survival data

Summary Cell survival was studied by analyzing the inactivation probability density function and its fundamental parameters. Mean , variance ² and modeD _mode were evaluated and a set of equations relating these parameters to the usual parameters of the multitarget, multihit and linear-quadratic modelsD _o andn, and, k and are reported. The multihit equation used was an extension of the usual equation, to allow parameterk to assume values that are not necessarily integers. In the multitarget curve, the mode of inactivation probability density function, proved to be the quasi-threshold doseD _q =D _o ln(n). Relative variance, degree of asymmetry and degree of peakedness can be calculated from the shape parametersn in the multitarget model,k in the multihit model, and in the linear-quadratic model. From an analysis of eight published cell survival sets of data, on C3H10T1/2 cells exposed to low LET radiations, it was found that ,, andSF ₂ are the parameters which exhibit the least variation from experiment to experiment and the least variation in selecting the range of data available for estimation.     

Chromosome numbers, genome sizes, cell volumes and evolution of snake-head fish (family Channidae)

Chromosome number, C-value and cell volume studies were carried out on three species of the genus Channa , viz., C. punctatus, C. striatus and C. gachua . The chromosome number, karyotypic structure and DNA content per cell along with cell volume are reported and described. A series of chromosomal rearrangements are established in three different karyotypes along with polyploidy. Both pericentric inversion and Robertsonian fusion played a major role in chromosome rearrangements. The nuclear DNA content of these three species is within 19-29% of the present-day placental mammals, and is thus lower than the median amount for fishes in general and teleosts in particular. Their lower DNA content suggests that the three species of the family Channidae are highly specialized, and this is supported by their known morphologic, reproductive, behavioural and ecological characteristics.
The evolutionary significance of these chromosomal rearrangements, their origin and their mode of establishment are discussed. A probable phylogenetic model based on karyotype, C-value and chromosomal rearrangements of the genus is presented.     

Graph-based methods for analysing networks in cell biology

Availability of large-scale experimental data for cell biology is enabling computational methods to systematically model the behaviour of cellular networks. This review surveys the recent advances in the field of graph-driven methods for analysing complex cellular networks. The methods are outlined on three levels of increasing complexity, ranging from methods that can characterize global or local structural properties of networks to methods that can detect groups of interconnected nodes, called motifs or clusters, potentially involved in common elementary biological functions. We also briefly summarize recent approaches to data integration and network inference through graph-based formalisms. Finally, we highlight some challenges in the field and offer our personal view of the key future trends and developments in graph-based analysis of large-scale datasets.     

ESSA: an integrated and interactive computer tool for analysing RNA secondary structure.

With ESSA, we propose an approach of RNA secondary structure analysis based on extensive viewing within a friendly graphical interface. This computer program is organized around the display of folding models produced by two complementary methods suitable to draw long RNA molecules. Any feature of interest can be managed directly on the display and highlighted by a rich combination of colours and symbols with emphasis given to structural probe accessibilities. ESSA also includes a word searching procedure allowing easy visual identification of structural features even complex and degenerated. Analysis functions make it possible to calculate the thermodynamic stability of any part of a folding using several models and compare homologous aligned RNA both in primary and secondary structure. The predictive capacities of ESSA which brings together the experimental, thermodynamic and comparative methods, are increased by coupling it with a program dedicated to RNA folding prediction based on constraints management and propagation. The potentialities of ESSA are illustrated by the identification of a possible tertiary motif in the LSU rRNA and the visualization of a pseudoknot in S15 mRNA.     

Enzyme microelectrophoresis of cell colonies

Mouse A9 cell colonies containing 1 000–2 000 cells can be electrophoresed in thin starch gels and enzyme activity can be detected for phosphohexose isomerase (PHI), malate dehydrogenase (MDH), lactate dehydrogenase (LDH), adenylate kinase (AK) and nucleoside phosphorylase (NP). The new methods developed here are techniques of manual micromanipulation of the cell colony samples and slight modifications of routine methods of starch gel electrophoresis.     

Raman spectroscopy and WAXS method as a tool for analysing ion-exchange properties of alginate hydrogels

Hydrogels are cross-linked three-dimensional macromolecular networks that contain a large fraction of water within their structure. One of the most important properties of alginate hydrogels, leading to their broad versatility, is their ability for controlled uptake, release and retention of molecules. This ability, in turn, is due to specific interactions of the macromolecular network with the diffusing or retained molecule. Raman spectroscopy has been employed to characterize the diffusion properties of solutes in hydrogels. Besides their application in the food sector, they are used in many biomedical, pharmaceutical and technical areas; for example, as a natural tissue or drug carriers. In the latter case, controlled release of drugs from a wound dressing is of particular interest-or ion exchange between the drug and the structure of the dressing. Raman active vibrations were used to show the areas responsible for the penetration of the model azo-dyes (based on non-genotoxic benzidine analogs) within Ca-alginate/carboxymethylcellulose Medisorb A wound dressing. In this case, the intensity of the stretching bands was used to obtain the concentration profiles of the model dye in alginate/carboxymethylcellulose gel (Medisorb A). The characteristic band at 1511 cm(-1) indicates that new band positions were observed following dye adsorption on wound dressing. The Raman spectra of alginate immersed for different times in Ringer's solution reveal peak shifts. Differences in peak shapes and the appearance of new bands are observed as the sodium content increased. Raman spectra give direct information on the exchange process. There are also new peaks appearing at 1034-1016 and 850 cm(-1) regions in the spectra after the release studies. This could, therefore, correspond to a partial bonding between sodium and oxygen atoms (the guluronic units originate a band at approximately 1025 cm(-1)). The aim of the examination in this paper also was to investigate the crystallinity index of Medisorb A wound dressing dyed (or undyed) and Medisorb A wound dressing after the release process in Ringer's solution (the crystallinity index is about 65%). In WAXS curves we can observed additional peaks (2theta at 32 degrees and 45 degrees ).     

Papillation in Bacillus anthracis colonies: a tool for finding new mutators

Colonies of Bacillus anthracis Sterne allow the growth of papillation after 6 days of incubation at 30°C on Luria–Bertani medium. The papillae are due to mutations that allow the cells to overcome the barriers to continued growth. Cells isolated from papillae display two distinct gross phenotypes (group A and group B). We determined that group A mutants have mutations in the nprR gene including frameshifts, deletions, duplications and base substitutions. We used papillation as a tool for finding new mutators as the mutators generate elevated levels of papillation. We discovered that disruption of yycJ or recJ leads to a spontaneous mutator phenotype. We defined the nprR/papillation system as a new mutational analysis system for B. anthracis. The mutational specificity of the new mutator yycJ is similar to that of mismatch repair‐deficient strains (MMR^‐) such as those with mutations in mutL or mutS. Deficiency in recJ results in a unique specificity, generating only tandem duplications.     

SNP-minisequencing as an excellent tool for analysing degraded DNA recovered from archival tissues

SNP-minisequencing has become common in forensic genetics, especially for analysing degraded or low copy number DNA (LCN DNA). The aim of this study was to examine the usefulness of five SNP (single nucleotide polymorphism) markers for analyzing degraded and LCN DNA recovered from archival samples. DNA extractions of eight formalin-fixed paraffin-embedded (FFPE) tissues were performed and DNA fragments were amplified in one multiplex PCR (polymerase chain reaction). SNPs were identified in a minisequencing reaction and a gel electrophoresis in ABI Prism 377 Sequencer. The research confirmed the usefulness of SNP-minisequencing for analysing FFPE tissues.     

Review: An integrated graphical tool for analysing impacts and services provided by livestock farming

Livestock farming is criticized for negatively impacting the environment, concerns about animal welfare and the impact of excessive meat consumption on human health. However, livestock farming provides other underappreciated and poorly communicated benefits to society in terms of employment, product quality, cultural landscapes and carbon storage by grasslands. Few attempts have been made so far to simultaneously consider the services and impacts provided by livestock production. Here, we propose an integrated graphical tool, called the ‘barn’ to explicitly summarize the synergies and trade-offs between services and impacts provided by livestock farming. It illustrates livestock farming interacting with its physical, economic and social environment along five interfaces: (i) Markets, (ii) Work and employment, (iii) Inputs, (iv) Environment and climate, (v) Social and cultural factors. This graphical tool was then applied by comparing two contrasting livestock production areas (high livestock density v. grassland-based), and the dominant v. a niche system within a crop-livestock area. We showed the barn could be used for cross-comparisons of services and impacts across livestock production areas, and for multi-level analysis of services and impacts of livestock farming within a given area. The barn graphically summarizes the ecological and socio-economic aspects of livestock farming by explicitly representing multiple services and impacts of different systems in a simple yet informative way. Information for the five interfaces relies on available quantitative assessments from the literature or data sets, and on expert-knowledge for more qualitative factors, such as social and cultural ones. The ‘barn’ can also inform local stakeholders or policy-makers about potential opportunities and threats to the future of livestock farming in specific production areas. It has already been used as a pedagogical tool for teaching the diversity of services and impacts of livestock systems across Europe and is currently developed as a serious game for encouraging knowledge exchange and sharing different viewpoints between stakeholders.     

Calibur: a tool for clustering large numbers of protein decoys

Background  

Ab initio protein structure prediction methods generate numerous structural candidates, which are referred to as decoys. The decoy with the most number of neighbors of up to a threshold distance is typically identified as the most representative decoy. However, the clustering of decoys needed for this criterion involves computations with runtimes that are at best quadratic in the number of decoys. As a result currently there is no tool that is designed to exactly cluster very large numbers of decoys, thus creating a bottleneck in the analysis.     

Improved protocols for the isolation and in-situ cryopreservation of cell colonies

Stable transfection and cloning of cells often require physical separation of cell colonies. In order to conveniently isolate cell clones from petri dishes, we developed a protocol starting with a soft agar overlay of cells. This reduces the risk of cell diffusion between different colonies. Cells from individual colonies are mechanically removed, incubated with trypsin, and cell suspensions are seeded onto parallel microtiter plates. The cell clones on one microtiter plate can be cryopreserved in situ using the protocol described here which was tested for a variety of cell lines. Replica plates can be used for screening and further expansion of interesting clones. If screening can also be performed in situ, e.g., by immunocytochemistry, immunofluorescence, or the polymerase chain reaction, it is possible to perform most steps necessary in cell cloning experiments on microtiter plates.     

Litter sizes of Ha(ICR) mice: a preference for even numbers?

The frequency distributions of litter sizes at birth for eight sublines derived from the same Ha(ICR) mouse stock with or without selection for large litters have their modes at even numbers of young. Each of the eight sublines produced a majority of even-numbered litters. An upward integral shift relationship among the medians of the frequency distributions was established. These findings are interpreted to mean that, in response to continued plus selection, ovarian and uterine performance of the Ha(ICR) bipartite reproductive system tends to be maximal bilaterally, which results in an excess of even-numbered litter sizes.     

Analysis of pure and mixed murine mast cell colonies

Mast cells have been proposed to originate from diverse sources, including connective tissues, macrophages, T lymphocytes, and hemopoietic cells. Evidence for a hemopoietic origin of mast cells includes the presence of mast cell precursors in spleen colonies and the presence of mast cells in hemopoietic colonies in culture. Here we report a detailed analysis of mouse spleen mixed hemopoietic colonies containing mast cells. All of the colonies in cultures plated at low cell densities were individually removed for analysis by May-Grunwald-Giemsa staining on day 15 of culture. Examination of five dishes which contained a total of 82 colonies showed 16 pure mast cell colonies and 36 mixed mast cell colonies. Sixteen different combinations of cell types were seen and were not distinguishable from each other in situ. The most diverse type of mixed colony contained macrophages (m), neutrophils (n), eosinophils (e), mast cells (Mast), megakaryocytes (M), erythroid cells (E), and blast cells. The clonal origin of mixed mast cell colonies was established by the replating of single cells obtained from blast cell colonies. Individual cells were removed with a micromanipulator, replated, and allowed to grow for 15 days. Cytospin preparations of 10 such colonies showed diverse combinations of cell lineages which were seen in the different types of mixed mast cell colonies described above. Replating studies of mixed mast cell colonies were carried out and a high incidence of replating was seen. Approximately one half of these colonies formed only mast cell colonies upon replating. Further studies showed that pure mast cell colonies could be serially replated four to five times. The replating efficiency of cells in the primary mast cell colonies varied over a wide range (2.5–44%) with an average replating efficiency of 13%. The data also revealed that cells containing metachromatic granules possess significant proliferative capacity. From these studies of pure and mixed mast cell colonies, we concluded (1) that mast cells are in wide variety of types of mixed colonies and that the in situ identification of mixed colonies is unreliable, (2) that mast cells are derived from pluripotent hemopoietic stem cells, and (3) that mast cells with metachromatic granules can have a high proliferating ability.     

An improved method for staining cell colonies in clonogenic assays

Clonogenic assay is a widely used experimental approach to test for the effects of drugs/genes on the growth and proliferative characteristics of cells in vitro. Accurate quantitation of treatment effects in clonogeneic assays depends on the ability to visualize and count cell colonies precisely. We report a novel method (referred as ETeB) for staining cell colonies grown on plastic and specially coated substrates like collagen. Using colon cancer cell lines grown on plastic and collagen, we compared the colony staining efficiencies of the widely used methylene blue, and Ethidium bromide (ETeB) stains. Results show that the ETeB protocol works well on plastic and is extremely effective for staining colonies on collagen when compared to methylene blue. The key features and advantages of ETeB technique are; (a) reduction in background for colonies grown on collagen and possibly other substrates, (b) the whole procedure takes less than a minute, (c) no post-stain washing step is required which eliminates colony losses for cell lines that are loosely adherent, (d) colony visualization and counting can be done immediately following the staining procedure using a standard UV illuminator and software, and (e) the method works across a wide variety of cell lines. The simplicity and robustness of this procedure should warrant its usage in both small and large-scale clonogenic experiments.

DNA vaccines as a tool for analysing the protective immune response against rhabdoviruses in rainbow trout

DNA vaccines based on the glycoprotein genes of the salmonid rhabdoviruses VHSV and IHNV have been demonstrated to be very efficient in inducing a protective immune response against the respective diseases in rainbow trout. Nanogram doses of plasmid DNA delivered by intramuscular injection are sufficient to induce high levels of immunity in fingerling-size fish, whereas larger fish require more vaccine for protection. The protection is long lasting and, more surprisingly, is partly established already 4 days post vaccination. The early protection involves cross-protective anti-viral defence mechanisms, while the long duration immunity is highly specific. The nature of these immune response mechanisms is discussed and it is suggested that the efficacy of the vaccines is related to their ability to activate the innate immune system as it is activated by live virus.     

Kinetic analysis of megakaryocyte numbers and ploidy levels in developing colonies from mouse bone marrow cells

Abstract. The kinetics of megakaryocyte formation from mouse bone marrow cells in semi-solid medium was studied directly in the culture dish by staining the cells for acetylcholinesterase after drying the cultures. A WEHI-3 cell-conditioned medium (WEHI-3 CM) was used as a general source of stimulus for megakaryocyte colony formation. The addition of peritoneal exudate supernatant as well as WEHI-3 CM increased the frequency of megakaryocyte colonies detected. Colonies containing acetylcholinesterase-positive cells were first detected at day 3. Maximum numbers of 25–40 megakaryocyte colonies per 10⁵ nucleaet mouse bone marrow cells were observed from days 7 to 11. The mean number of cells within each colony increased progressively with time of culture, and a modal range of 11–20 cells was obtained by day 7. Between 3 and 200 cells per colony were generally detected. A continuous distribution of the number of megakaryocytes per colony suggests that the clonable precursor cells are not synchronized either with respect to maturation stage or with respect to their capability to undergo nuclear endoreduplication. The addition of peritoneal exudate supernatant to the cell cultures increased the DNA levels of megakaryocytes grown in the presence of WEHI-3 CM but did not affect the number of cells per colony. The DNA content of colony megakaryocytes was measured after staining the cells with Feulgen reagent. A modal DNA value of 8 N was observed between days 4 and 7 for megakaryocytes stimulated with WEHI-3 CM. In the presence of both WEHI-3 CM and peritoneal exudate supernatant, the DNA content of megakaryocytes increased with the time of cell culture. Modal DNA values increased from 8 N at days 4 and 5, to 16 N by day 6. In these optimally stimulated cultures, 44% of colony megakaryocytes were 32 N or greater, a proportion higher than in normal bone marrow, but similar to that seen in the marrow of acutely thrombocytopenic animals. It is concluded that megakaryocytopoiesis in cell cultures is not a strictly controlled process with respect to cell division and endomitosis and that when certain culture conditions are employed, megakaryocyte development in vitro might reflect that seen in a stressed animal condition.