Computer-based image analysis for the automated counting and morphological description of microalgae in culture

A largely unexplored area is the application of digital image processing to counting and sizing of microalgal cells from culture. Commercial systems are available, but have not been tested, nor necessarily optimized for high speed counting and sizing of phytoplankton. The present work describes the design, construction, specifications and comparative performance of an inexpensive system optimized for counting and sizing microalgal cells. This system has been tested with cells of the picoplankton to nanoplankton size ranges (1–20 μm). The hardware was a widely available standard microcomputer, an inexpensive video camera and monitor, and a video digitization board (frame grabber). A modifiable menu-driven program (PHYCOUNT) was written and provisions made to make this program available to other workers. The program is constructed such that it can be adapted to a variety of hardware setups Video digitization boards). Comparison of growth curves for microagae revealed there were no significant differences in division rate and cell yield as assessed by the image analysis method compared to manual counts with a hemacytometer. Several hundred cells were counted routinely within 10–15 s, far exceeding the counting rate achieved by hand tally. A variable transect feature allowed sampling every nth pixel and provided a substantial increase in execution speed. More than 1000 counts can be done per day. A protocol for the use of 96-well plates of polyvinyl chloride as counting chambers contributed to the processing of large numbers of samples rapidly. Other routines developed provided subtended area, defined the coordinates of cell perimeter, and derived cell length and width. The calculation of the latter two parameters was usually done off-line as data output is in standard numerical form accessible by other programs. Experience with daily use of the PHYCOUNT program and imaging hardware reveal that the system is reliable for cell counting and sizing. The presence of bacteria in the algal cultures does not affect cell counting or sizing.     

BIOSITE: a program for the interactive comparison of aligned homologous protein sequences

A program, BIOSITE, providing for the interactive visual comparisonof aligned homologous amino-acid sequences is presented, includingan example of its application. The program allows for two typesof comparison sequence to be generated: an ‘identity’sequence and a ‘difference’ sequence. These maybe used on subsets of sequences and in further comparisons toidentify candidate sites involved in a distinct functional property.The program should prove a useful tool for biologists engagedin understanding sequence—function relationships.     

Olga--oligonucleotide primer design program for the Atari ST

A program to facilitate the design of oligonucleotide primnershas been devised. Olga is written in draft ANSI standard ‘C’and makes use of the implementation of Digital Research GEM(Graphics Environment Manager) on the Atari ST Olga is specificallysuited to the polymnerase chain reaction (PCR) allowing si analysisof two primer sequences. The advantage of Olga is that it providesin one program analyses for direct repeats, secondary structuresand primer dimerization as well as several useful ‘finishing’tools for workers engaged in PCR optimization and oligonucleotidesyntheses.     

Optimal alignments of biological sequences on a microcomputer

An algorithm and a program have been developed which enableoptimal alignments of biological sequences on an 8–bitmicrocomputer. The compiled program can process sequences upto 1000 residues on a Commodore 64. Since this program was writtenoriginally in the BASIC language, it may readily be adaptedto other microcomputers with small changes. Received on March 11, 1985; accepted on March 14, 1985     

An evaluation of antibiotics for the elimination of Agrobacterium tumefaciens from walnut somatic embryos and for the effects on the proliferation of somatic embryos and regeneration of transgenic plants

Four antibiotics were evaluated for their effects on eliminating the hypervirulent Agrobacterium tumefaciens strain C58C1 ATHV Rif^R (pEHA101)/p35-gus-intron from walnut somatic embryos and on the production of secondary somatic embryos and the transformed somatic embryos. Exposure to 100–1000 mg l⁻¹ of ampicillin, carbenicillin or cefotaxime respectively for up to 60 days did not eliminate the A. tumefaciens while timentin at 500–1000 mg l⁻¹ eradicated it from somatic embryos. One-hour acidified medium treatments and the addition of 100 mg l⁻¹ kanamycin to 500 mg l⁻¹ ampicillin, carbenicillin, cefotaxime or timentin were of little help in eliminating the Agrobacterium. All four antibiotics reduced somatic embryo production, carbenicillin minimally and cefotaxime maximally, especially at higher concentrations, in comparison with antibiotic-free medium. Putative transformed embryos were selected for continued proliferation on a 100 mg l⁻¹ kanamycin-containing medium. Histochemical assessments indicated that more gus-positive somatic embryos, particularly fully gus-positive embryos, regenerated from timentin-containing medium than from other antibiotic-containing media under equivalent conditions. Transformed embryos have been grown and converted into plants and gus activity was observed in whole plants. Received: 13 July 1999 / Revision received: 2 December 1999 / Accepted: 6 December 1999     

A versatile approach for modeling and simulating the tacticity of polymers

We are introducing a versatile computerized approach to model and simulate polymer tacticities using seven single-stage statistical models. The theory behind the models, e.g., Bovey’s versus Price’s, Bernoullian, 1st or 2nd order Markovian, enantiomeric types, and combinations thereof is explained. One of the models, "E-B gen", which can be used to produce four types of enantiomorphically controlled tacticities, and the pentad distribution for the model "E-M1" are reported here for the first time. The relations of chain-end controlled models to binary copolymerizations are discussed in detail, and equations for the conversion of tacticity based probabilities to reactivity ratios to obtain related n-ad distributions are presented. The models were applied to 20 polymers with exemplary tacticities found in the literature. A related software program (“Polytact”) based on Microsoft’s Excel has been designed to calculate all relevant characteristics of the polymer tacticity and to present them in graphical form in a user-friendly manner. The program can be used to produce graphs of the triad, pentad and sequence length distributions and a simulation of 50 monomer repeat units in the polymer for each of the seven models. One of the main intended uses of the program is to compare the computed n-ad distributions to those of experimental polymers obtained from NMR spectroscopy and to gain insight into the polymerization mechanisms.     

Software for the development and evaluation of probabilistic identification matrices

A number of programs are described for the development and evaluationof probabilistic identification matrices for use with computer-assistedidentification. The program BEST reads an initial matrix ofper cent probabilities for all the binary characters examinedduring a cluster analysis and determines the most useful setof tests for distinguishing taxa in the matrix. Program RESORTcreates from the initial matrix an identification matrix inwhich the order of the tests may be different or the numberof tests reduced. A printed version of a matrix can be producedby MATPRJNT, which creates tables giving the per cent probabilityof a positive test result and the test results presented as‘–’, ‘v’ and ‘+’ dependingon a user-specified threshold. Program IDSC evaluates an identificationmatrix by calculating the best identification score for eachtaxon in the matrix using the expected result for each test.The programs were written in FORTRAN 77 and can be run on anymicrocomputer using the PC/MS-DOS operating system. Received on April 27, 1990; accepted on November 5, 1990     

An interactive graphic program for calculating the secondary structure content of proteins from circular dichroism spectrum

A graphic program has been developed to calculate the secondarystructure content of proteins from their circular dichroismspectrum. All the information concerning analysis and resultsare given on a single screen. The actual and the theoreticalspectra are plotted to allow visual inspection of the fit quality.The percentages of secondary structure and statistical parameters(r. m. s., residuals) are provided. The program is fully interactivefor spectra analysis. Moreover, cursors driven by a mouse orarrow keys are moveable onto spectra yielding all the informationconcerning a given wavelength, such as the theoretical and experimentalellipticities, wavelength, values of reference model for -helix,ß-sheet and ß-turn. Interfaces are providedfor the CONTIN program ofProvencher and Glockner.     

PROANAL version 2: multifunctional Program for analysis of multiple protein sequence alignments and for studying the structure-activity relationships in protein families

A new version of the program PROANAL is described. A multiplelinear regression analysis of the protein structure–activityrelationship allows one to investigate the combina–tionsof protein sites and factors influencing the activity. The programalso provides the possibility to seek out protein sites, conservativeor variable in variations of physico–chemical characteristics,and regions with high or low values of these characteristics.PROANAL2 may be useful in the simulation of protein–engineeringexperiments and in the search of a number of protein regionssuch as functional sites, secondary structures, solvent-exposedregions, T– and B–cell antigenic determinants, etc.     

Injectable Polysaccharide Microcapsules for Prolonged Release of Minocycline for the Treatment of Periodontitis

Injectable polysaccharide microcapsules holding minocycline were fabricated from alginate and chitosan for the treatment of periodontitis. The microcapsules were examined for the release and degradation of minocycline, as well as antimicrobial activity. The microcapsules were biodegradable and released minocycline between 10 and 1000 μg ml⁻¹, which was higher than the usual therapeutic concentration (1–5 μg ml⁻¹), for up to 7 days. These microcapsules showed a statistically significant suppression of pathogenic bacteria, such as Prevotella intermedia causing periodontitis. The microcapsules are thus potentially useful for drug delivery for the treatment of periodontitis.     

Comparing machine-independent versus machine-specific parallelization of a software platform for biological sequence comparison

A platform program that performs biological sequence comparisonprovides a case study to compare the relative advantages ofa machine–independent approach to parallel computationversus a machine-specific approach. The program consists oftwo routines: (i) PSCANLIB, which compares a single biologicalsequence against a database of sequences, and (ii) PCOMPLJB,which compares a database of sequences against another databaseof sequences, or against itself. The program was first parallelizedto run on the Intel Hypercube parallel computer using nativeHypercube commands to coordinate the parallel computation. Theparallelization logic of the program was then translated intoa machine–independent parallel programming language, Linda.Tliese two approaches to parallelization are contrasted in termsof: (i) the expressive power of the logic that coordinates theparallel computation, (ii) the portability of the machine–independentversion to other parallel machines and (Hi) the relative efficiencyof the two versions of the program. In the benchmark tests reported,the benefits of the machine–independent approach wereachieved with only a modest sacrifice in efficiency.     

A general purpose non-linear curve fitting program for the British Broadcasting Corporation Microcomputer

Software for non–linear curve fitting has been writtenin BASIC to execute on the British Broadcasting CorporationMicrocomputer. The program uses the direct search algorithmPattern–search, a robust algorithm that has the additionaladvantage of needing specification of the Junction without inclusionof the partial derivatives. Although less efficient than gradientmethods, the program can be readily configured to solve low–dimensionaloptimization problems that are normally encountered in lifesciences. In writing the software, emphasis has been placedupon the ‘user interface’ and making the most efficientuse of the facilities provided by the minimal configurationof this system. Received on March 4, 1985; accepted on March 14, 1985     

Influence of the pycnocline on the vertical migration of chaetognaths in the northern Benguela

The vertical distribution and migration patterns of chaetognathswere studied at a sampling station off northern Namibia (18°00'S,10°30'E during a 48 h sampling cycle. The sampling areawas characterized by mixing of the Angola current with the northernmostwaters of the Benguela current in the surface region. The continuousflow of the Angola current during the study period gave riseto a thermocline and a halocline at a depth of 40 m. The samplingintervals used to study the behaviour of epipelagic chaetognathspecies under these conditions were 200–100, 100–60,60–40, 40–20 and 20–0 m. A total of 10 differentchaetognath species were captured. Sagitia enflata was the predominantspecies, with a mean density of 4400 individuals per 1000 m³in daytime hauls, accounting for 54.1% of all individuals collectedin the samples. Sagitia minima was the next most predominantspecies, with a mean density of 2400 individuals per 1000 m³in daytime hauls, accounting for 16.6% of the chaetognath communitysampled. Three maturity stages were considered in analysingpossible ontogenetic migrations. Nearly all the species wereaggregated above the pycnocline, and stages I and II of manyspecies carried out short migrations in the surface layers.The limited migration pattern, together with a staggered distributionof the different species and stages above the pycnocline, hasbeen interpreted as a space partitioning mechanism to preventintra- and interspecific competition.     

Thermal denaturation of double-stranded nucleic acids: prediction of temperatures critical for gradient gel electrophoresis and polymerase chain reaction.

A program is described which calculates the thermal stability and the denaturation behaviour of double-stranded DNAs and RNAs up to a length of 1000 base pairs. The algorithm is based on recursive generation of conditional and a priori probabilities for base stacking. Output of the program may be compared directly to experimental results; thus the program may be used to optimize the nucleic acid fragments, the primers and the experimental conditions prior to experiments like polymerase chain reactions, temperature-gradient gel electrophoresis, denaturing-gradient gel electrophoresis and hybridizations. The program is available in three versions; the first version runs interactively on VAXstations producing graphics output directly, the second is implemented as part of the HUSAR package at GENIUSnet, the third runs on any computer producing text output which serves as input to available graphics programs.     

A program for drawing evolutionary trees

From the measures of evolutionary distance between pairs ofsequences in a set, it is possible to infer the genetic treeor trees which best fit these known data. DENDRON is a new program,written in FORTRAN 66, which computes an initial tree from thebottom-up, then searches among increasingly divergent treesfor a better fit. As a check on the consistency of the measures,the program tests all triplets for the triangle inequality.DENDRON also calculates a single ‘top-down’ tree,progressing from the trunk to the twigs, for comparison withthe ‘bottom-up’ trees. Received on August 17, 1987; accepted on June 1, 1988     

POEMS: program for outliers elimination in multidimensional space

A Program for Outliers Elimination in Multidimensional Space(POEMS), which allows the user to eliminate outliers from trainingspaces as a prior step to any statistical study, is presented.Even though the program can be applied to any scientific field,the characteristics of POEMS makes it particularly suitablefor ‘series design’ on Quantitative Structure -Activity Relationships studies. Received on March 1, 1988; accepted on May 3, 1988     

APL program for evaluation of heat processes for spherical foods

The present program solves the heat transfer equations for sphericalfoods by a numerical procedure that uses the Crank–Nicolsonmethod. The program is implemented for conditions with as fewrestrictions as possible to enhance versatility. The resultsagree with the analytical solutions for two particular casesof boundary conditions. The program also computes the thermaleffect in the foodstuff from the temperature data obtained bythe numerical program.     

Stable Isotopic and Trace Elemental Compositions of Otoliths and the Stock Structure of Pacific Cod, Gadus macrocephalus

Synopsis We examined the stock composition and life history of Pacific cod, Gadus macrocephalus, in the northeast Pacific Ocean from sagittal otoliths collected from three marine fishing areas in Washington State, U.S.A. We analyzed both stable isotope ratios (δ¹⁸O and δ¹³C) and trace elemental concentrations (Sr, Mg, Na, Fe, Mn) for these otoliths. The combination of δ¹⁸O and δ¹³C, and correlation of δ¹⁸O vs. 1000Sr/Ca and 1000Mg/Ca showed clear separations between North Puget Sound and coastal cod, suggesting there might be two different spawning stocks in the region. The North Puget Sound cod might represent an ‘Estuary-type’ from the Strait of Georgia, whereas coastal cod might represent an ‘Ocean-type’ from the Pacific west coast. Isotopic variations from five representative otoliths also showed a two-stage life history for Pacific cod, and a critical transition period for cod in migration to the ocean or in age of sexual maturity. These chemical interpretations and conclusions appear in agreement with biological observations of Pacific cod, and are consistent with results of previous studies for other marine fish species in nearby areas.     

A program for the graphic representation and manipulation of DNA sequences

We have developed a program for the graphic representation andmanipulation of DNA sequences. The program (named CARTE fromthe French for ‘map’) is intended as a tool in theplanning and analysis of recombinant DNA experiments. DNA sequencesare represented as standard restriction maps, using any desiredcombination of restriction enzymes. Features of interest, suchas promoters or coding sequences, can be highlighted. The sequencecan be manipulated to mimic cloning, using deletions, insertionsor replacements at specified sites. This process is facilitatedby the simultaneous display of a graphic map of the entire sequence,a detailed picture of the work in progress, and a menu of functions. Received on November 17, 1986; accepted on March 12, 1987