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Photoassimilates such as sugars are transported through phloem sieve element cells in plants. Adapted for effective transport, sieve elements develop as enucleated living cells. We used electron microscope imaging and three-dimensional reconstruction to follow sieve element morphogenesis in Arabidopsis. We show that sieve element differentiation involves enucleation, in which the nuclear contents are released and degraded in the cytoplasm at the same time as other organelles are rearranged and the cytosol is degraded. These cellular reorganizations are orchestrated by the genetically redundant NAC domain-containing transcription factors, NAC45 and NAC86 (NAC45/86). Among the NAC45/86 targets, we identified a family of genes required for enucleation that encode proteins with nuclease domains. Thus, sieve elements differentiate through a specialized autolysis mechanism.

Despite the expression of the mutated gene in all muscles, selective muscles are involved in genetic muscular dystrophies. Different muscular dystrophies show characteristic patterns of fatty degenerative changes by muscle imaging, even to the extent that the patterns have been used for diagnostic purposes. However, the underlying molecular mechanisms explaining the selective involvement of muscles are not known. To test the hypothesis that different muscles may express variable amounts of different isoforms of muscle genes, we applied a custom-designed exon microarray containing probes for 57 muscle-specific genes to assay the transcriptional profiles in sets of human adult lower limb skeletal muscles. Quantitative real-time PCR and whole transcriptome sequencing were used to further analyze the results. Our results demonstrate significant variations in isoform and gene expression levels in anatomically different muscles. Comparison of the known patterns of selective involvement of certain muscles in two autosomal dominant titinopathies and one autosomal dominant myosinopathy, with the isoform and gene expression results, shows a correlation between the specific muscles involved and significant differences in the level of expression of the affected gene and exons in these same muscles compared with some other selected muscles. Our results suggest that differential expression levels of muscle genes and isoforms are one determinant in the selectivity of muscle involvement in muscular dystrophies.

We characterize allelic and gene expression variation between populations of the Glanville fritillary butterfly (Melitaea cinxia) from two fragmented and two continuous landscapes in northern Europe. The populations exhibit significant differences in their life history traits, e.g. butterflies from fragmented landscapes have higher flight metabolic rate and dispersal rate in the field, and higher larval growth rate, than butterflies from continuous landscapes. In fragmented landscapes, local populations are small and have a high risk of local extinction, and hence the long-term persistence at the landscape level is based on frequent re-colonization of vacant habitat patches, which is predicted to select for increased dispersal rate. Using RNA-seq data and a common garden experiment, we found that a large number of genes (1,841) were differentially expressed between the landscape types. Hexamerin genes, the expression of which has previously been shown to have high heritability and which correlate strongly with larval development time in the Glanville fritillary, had higher expression in fragmented than continuous landscapes. Genes that were more highly expressed in butterflies from newly-established than old local populations within a fragmented landscape were also more highly expressed, at the landscape level, in fragmented than continuous landscapes. This result suggests that recurrent extinctions and re-colonizations in fragmented landscapes select a for specific expression profile. Genes that were significantly up-regulated following an experimental flight treatment had higher basal expression in fragmented landscapes, indicating that these butterflies are genetically primed for frequent flight. Active flight causes oxidative stress, but butterflies from fragmented landscapes were more tolerant of hypoxia. We conclude that differences in gene expression between the landscape types reflect genomic adaptations to landscape fragmentation.

Insect flight is one of the most energetically demanding activities in the animal kingdom, yet for many insects flight is necessary for reproduction and foraging. Moreover, dispersal by flight is essential for the viability of species living in fragmented landscapes. Here, working on the Glanville fritillary butterfly (Melitaea cinxia), we use transcriptome sequencing to investigate gene expression changes caused by 15 min of flight in two contrasting populations and the two sexes. Male butterflies and individuals from a large metapopulation had significantly higher peak flight metabolic rate (FMR) than female butterflies and those from a small inbred population. In the pooled data, FMR was significantly positively correlated with genome-wide heterozygosity, a surrogate of individual inbreeding. The flight experiment changed the expression level of 1513 genes, including genes related to major energy metabolism pathways, ribosome biogenesis and RNA processing, and stress and immune responses. Males and butterflies from the population with high FMR had higher basal expression of genes related to energy metabolism, whereas females and butterflies from the small population with low FMR had higher expression of genes related to ribosome/RNA processing and immune response. Following the flight treatment, genes related to energy metabolism were generally down-regulated, while genes related to ribosome/RNA processing and immune response were up-regulated. These results suggest that common molecular mechanisms respond to flight and can influence differences in flight metabolic capacity between populations and sexes.

Previous studies have reported that chromosome synteny in Lepidoptera has been well conserved, yet the number of haploid chromosomes varies widely from 5 to 223. Here we report the genome (393 Mb) of the Glanville fritillary butterfly (Melitaea cinxia; Nymphalidae), a widely recognized model species in metapopulation biology and eco-evolutionary research, which has the putative ancestral karyotype of n=31. Using a phylogenetic analyses of Nymphalidae and of other Lepidoptera, combined with orthologue-level comparisons of chromosomes, we conclude that the ancestral lepidopteran karyotype has been n=31 for at least 140 My. We show that fusion chromosomes have retained the ancestral chromosome segments and very few rearrangements have occurred across the fusion sites. The same, shortest ancestral chromosomes have independently participated in fusion events in species with smaller karyotypes. The short chromosomes have higher rearrangement rate than long ones. These characteristics highlight distinctive features of the evolutionary dynamics of butterflies and moths., Marie Curie International Fellowship (PIOF-GA-2011-303312)

Profound understanding of the mechanisms foodborne pathogenic bacteria utilize in adaptation to the environmental stress they encounter during food processing and storage is of paramount importance in design of control measures. Chill temperature is a central control measure applied in minimally processed foods; however, data on the mechanisms the foodborne pathogen Clostridium botulinum activates upon cold stress are scarce. Transcriptomic analysis on the C. botulinum ATCC 3502 strain upon temperature downshift from 37°C to 15°C was performed to identify the cold-responsive gene set of this organism. Significant up- or down-regulation of 16 and 11 genes, respectively, was observed 1 h after the cold shock. At 5 h after the temperature downshift, 199 and 210 genes were up- or down-regulated, respectively. Thus, the relatively small gene set affected initially indicated a targeted acute response to cold shock, whereas extensive metabolic remodeling appeared to take place after prolonged exposure to cold. Genes related to fatty acid biosynthesis, oxidative stress response, and iron uptake and storage were induced, in addition to mechanisms previously characterized as cold-tolerance related in bacteria. Furthermore, several uncharacterized DNA-binding transcriptional regulator-encoding genes were induced, suggesting involvement of novel regulatory mechanisms in the cold shock response of C. botulinum. The role of such regulators, CBO0477 and CBO0558A, in cold tolerance of C. botulinum ATCC 3502 was demonstrated by deteriorated growth of related mutants at 17°C.

Studies on extracellular proteins (ECPs) contribute to understanding of the multifunctional nature of apoplast. Unlike vascular plants (tracheophytes), little information about ECPs is available from nonvascular plants, such as mosses (bryophytes). In this study, moss plants (Physcomitrella patens) were grown in liquid culture and treated with chitosan, a water-soluble form of chitin that occurs in cell walls of fungi and insects and elicits pathogen defense in plants. ECPs released to the culture medium were compared between chitosan-treated and nontreated control cultures using quantitative mass spectrometry (Orbitrap) and 2-DE-LC-MS/MS. Over 400 secreted proteins were detected, of which 70% were homologous to ECPs reported in tracheophyte secretomes. Bioinformatics analyses using SignalP and SecretomeP predicted classical signal peptides for secretion (37%) or leaderless secretion (27%) for most ECPs of P. patens, but secretion of the remaining proteins (36%) could not be predicted using bioinformatics. Cultures treated with chitosan contained 72 proteins not found in untreated controls, whereas 27 proteins found in controls were not detected in chitosan-treated cultures. Pathogen defense-related proteins dominated in the secretome of P. patens, as reported in tracheophytes. These results advance knowledge on protein secretomes of plants by providing a comprehensive account of ECPs of a bryophyte.

Kohonen‘s Self-Organizing Map (SOM) is combined with the Redundant Hash Addressing (RHA) principle. The SOM encodes the input feature vector sequence into the sequence of best-matching unit (BMU) indices and the RHA principle is then used to associate the BMU index sequence with the dictionary items. This provides a fast alternative for dynamic programming (DP) based methods for comparing and matching temporal sequences. Experiments include music retrieval and speech recognition. The separation of the classes can be improved by error-corrective learning. Comparisons to DP-based methods are presented.

The Self-Organizing Map (SOM) and Learning Vector Quantization (LVQ) algorithms are constructed in this work for variable-length and warped feature sequences. The novelty is to associate an entire feature vector sequence, instead of a single feature vector, as a model with each SOM node. Dynamic time warping is used to obtain time-normalized distances between sequences with different lengths. Starting with random initialization, ordered feature sequence maps then ensue, and Learning Vector Quantization can be used to fine tune the prototype sequences for optimal class separation. The resulting SOM models, the prototype sequences, can then be used for the recognition as well as synthesis of patterns. Good results have been obtained in speaker-independent speech recognition.

Yersinia pseudotuberculosis is an important pathogen that probably survives well in the modern food chain. However, little is known about the mechanisms that allow the growth of this pathogen in foods under stress conditions. The expression of rpoE encoding σ E was defined by quantitative real-time reverse transcription-PCR. Expression of rpoE was induced at 3°C, 37°C, and 42°C, under exposure to 3% NaCl, 3% ethanol, or high and low pH, in relation to its expression at the optimum growth temperature of 28°C of Y. pseudotuberculosis . Mutation of rpoE either impaired or abolished growth under stresses caused by low or high temperature, low pH, and ethanol. In addition, the growth temperature range of the mutant was significantly diminished compared to that of the wild-type strain IP32953. The results were confirmed with complementation of the mutant. Thus, σ E plays a significant role in the stress tolerance of Y. pseudotuberculosis IP32953 and probably contributes to the survival of this pathogen in the food chain.

In this study, we characterized a putative Flp/Tad pilus-encoding gene cluster, and we examined its regulation at the transcriptional level and its role in the virulence of potato pathogenic enterobacteria of the genus Pectobacterium. The Flp/Tad pilus-encoding gene clusters in Pectobacterium atrosepticum, Pectobacterium wasabiae and Pectobacterium aroidearum were compared to previously characterized flp/tad gene clusters, including that of the well-studied Flp/Tad pilus model organism Aggregatibacter actinomycetemcomitans, in which this pilus is a major virulence determinant. Comparative analyses revealed substantial protein sequence similarity and open reading frame synteny between the previously characterized flp/tad gene clusters and the cluster in Pectobacterium, suggesting that the predicted flp/tad gene cluster in Pectobacterium encodes a Flp/Tad pilus-like structure. We detected genes for a novel two-component system adjacent to the flp/tad gene cluster in Pectobacterium, and mutant analysis demonstrated that this system has a positive effect on the transcription of selected Flp/Tad pilus biogenesis genes, suggesting that this response regulator regulate the flp/tad gene cluster. Mutagenesis of either the predicted regulator gene or selected Flp/Tad pilus biogenesis genes had a significant impact on the maceration ability of the bacterial strains in potato tubers, indicating that the Flp/Tad pilus-encoding gene cluster represents a novel virulence determinant in Pectobacterium. Soft-rot enterobacteria in the genera Pectobacterium and Dickeya are of great agricultural importance, and an investigation of the virulence of these pathogens could facilitate improvements in agricultural practices, thus benefiting farmers, the potato industry and consumers.

The posttranscriptional regulator RsmA controls the production of plant cell wall degrading enzymes (PCWDE) and cell motility in the Pectobacterium genus of plant pathogens. In this study the physiological role of gene regulation by RsmA is under investigation. Disruption of rsmA gene of the Pectobacterium wasabiae strain, SCC3193 resulted in 3-fold decrease in growth rate and increased virulence. The comparison of mRNA levels of the rsmA(-) mutant and wild-type using a genome-wide microarray showed, that genes responsible for successful infection, i.e. virulence factors, motility, butanediol fermentation, various secretion systems etc. were up-regulated in the rsmA(-) strain. The rsmA(-) strain exhibited a higher propensity to swarm and produce PCWDE compared to the wild-type strain. Virulence experiments in potato tubers demonstrated that in spite of its more efficient tissue maceration, the rsmA(-) strain's ability to survive within the host is reduced and the infection site is taken over by resident bacteria. Taken together, in the absence of RsmA, cells revert to a constitutively infective phenotype characterized by expression of virulence factors and swarming. We hypothesize that lack of control over these costly energetic processes results in decreased growth rate and fitness. In addition, our findings suggest a relationship between swarming and virulence in plant pathogens.

Leuconostoc gasicomitatum is a psychrotrophic lactic acid bacterium (LAB) which causes spoilage in cold-stored modified-atmosphere-packaged (MAP) meat products. In addition to the fermentative metabolism, L. gasicomitatum is able to respire when exogenous heme and oxygen are available. In this study, we investigated the respiration effects on growth rate, biomass, gene expression, and volatile organic compound (VOC) production in laboratory media and pork loin. The meat samples were evaluated by a sensory panel every second or third day for 29 days. We observed that functional respiration increased the growth (rate and yield) of L. gasicomitatum in laboratory media with added heme and in situ meat with endogenous heme. Respiration increased enormously (up to 2,600-fold) the accumulation of acetoin and diacetyl, which are buttery off-odor compounds in meat. Our transcriptome analyses showed that the gene expression patterns were quite similar, irrespective of whether respiration was turned off by excluding heme from the medium or mutating the cydB gene, which is essential in the respiratory chain. The respiration-based growth of L. gasicomitatum in meat was obtained in terms of population development and subsequent development of sensory characteristics. Respiration is thus a key factor explaining why L. gasicomitatum is so well adapted in high-oxygen packed meat.

The role of the two-component system (TCS) CBO0366/CBO0365 in the cold shock response and growth of the mesophilic Clostridium botulinum ATCC 3502 at 15°C was demonstrated by induced expression of the TCS genes upon cold shock and impaired growth of the TCS mutants at 15°C.

Nebulin is a large actin-binding protein of the skeletal muscle sarcomere. Multiple isoforms of nebulin are produced from the 183-exon-containing nebulin gene (NEB). Mutations in NEB cause nemaline myopathy, distal myopathy, and core-rod myopathy.Nebulin mRNA expression was assessed by microarrays and RT-PCR in 21 human leg muscle and 2 brain samples. Protein expression was assessed by immunohistochemistry in 5 regions of 1 brain sample.Nebulin isoform diversity is as high in brain as in skeletal muscle. Isoforms with more than 22 super repeats seem to be more common than previously anticipated. Immunohistochemistry showed nebulin expression predominantly in the cytoplasm of pyramidal neurons but also in the cytoplasm of mainly subcortical endothelial cells.Nebulin, as in skeletal muscle, may have a role as an actin filament stabilizer or length regulator in neurons of the human brain, although patients with NEB mutations usually have normal cognition.

The expression of csdA , encoding an RNA helicase, was induced at 3°C in Yersinia pseudotuberculosis . The role of CsdA in Y. pseudotuberculosis under cold conditions was confirmed by impaired growth of insertional csdA mutants at 3°C. The results suggest that CsdA is crucial for Y. pseudotuberculosis survival in the chilled food chain.

Yersinia pseudotuberculosis is a significant psychrotrophic food pathogen whose cold tolerance mechanisms are poorly understood. Signal transduction systems serve to monitor the environment, but no systematic investigation of their role at cold temperatures in Y. pseudotuberculosis has yet been undertaken. The relative expression levels of 54 genes predicted to encode proteins belonging to signal transduction systems in Y. pseudotuberculosis IP32953 were determined at 28°C and 3°C by quantitative real-time reverse transcription-PCR. The relative expression levels of 44 genes were significantly (p

While temperature-dependent induction of flagella is a well-characterized phenomenon in Listeria monocytogenes, the essentiality of increased flagellum production during growth at low temperatures remains unclear. To study this relationship, we compared the relative expression levels of two motility genes, flhA and motA, at 3°C, 25°C and 37°C in L. monocytogenes strain EGD-e by using qRT-PCR, and compared the growth curves, motility, and flagellation between the wild-type and flhA and motA deletion mutants. The relative expression levels of flhA and motA at 3°C were significantly higher than at 37°C (p

SUMMARY The areas of the pathogenicity island (PAI) designated as ‘colonization region’ (CR) and ‘toxicogenic region’ (TR) [Lerat et al. (2009) Mol. Plant Pathol. 10, 579–585] contain genes required for virulence and phytoxin production, respectively, in Streptomyces spp. causing common scab on potatoes. The PAI was tested for genetic variability by microarray analysis in strains of S. turgidiscabies isolated from potatoes in Finland. The data revealed four types of PAI based on divergent CR and TR which occurred in different combinations. Only one PAI type was highly similar to S. scabies (strains 87.22 and ATTC49173). Using probes designed for the predicted genes of S. scabies, two gene clusters in S. scabies appeared to be similar to most strains of S. turgidiscabies and contained PAI genes corresponding to CR and TR. They were located approximately 5 Mb apart in the S. scabies genome, as compared with only 0.3 Mb in S. turgidiscabies Car8. Data from comparative genomic hybridization with probes designed for S. scabies genes and for the PAI of S. turgidiscabies were compared by multilocus cluster analysis, which revealed two strains of S. turgidiscabies that were very closely related at the whole-genome level, but contained distinctly different PAIs. The type strain of S. reticuliscabiei (DSM41804; synonymous to S. turgidiscabies) was clustered with S. turgidiscabies. Taken together, the data indicate wide genetic variability of PAIs among strains of S. turgidiscabies, and demonstrate that PAI is made up of a mosaic of regions which may undergo independent evolution.

Long-term effects of elevated CO(2) and O(3) concentrations on gene expression in silver birch (Betula pendula Roth) leaves were studied during the end of the growing season. Two birch genotypes, clones 4 and 80, with different ozone growth responses, were exposed to 2x ambient CO(2) and/or O(3) in open-top chambers (OTCs). Microarray analyses were performed after 2 years of exposure, and the transcriptional profiles were compared to key physiological characteristics during leaf senescence. There were genotypic differences in the responses to CO(2) and O(3). Clone 80 exhibited greater transcriptional response and capacity to alter metabolism, resulting in better stress tolerance. The gene expression patterns of birch leaves indicated contrasting responses of senescence-related genes to elevated CO(2) and O(3). Elevated CO(2) delayed leaf senescence and reduced associated transcriptional changes, whereas elevated O(3) advanced leaf senescence because of increased oxidative stress. The combined treatment demonstrated that elevated CO(2) only temporarily alleviated the negative effects of O(3). Gene expression data alone were insufficient to explain the O(3) response in birch, and additional physiological and biochemical data were required to understand the true O(3) sensitivity of these clones.

Comparative genomic hybridization analysis of 32 Nordic group I Clostridium botulinum type B strains isolated from various sources revealed two homogeneous clusters, clusters BI and BII. The type B strains differed from reference strain ATCC 3502 by 413 coding sequence (CDS) probes, sharing 88% of all the ATCC 3502 genes represented on the microarray. The two Nordic type B clusters differed from each other by their response to 145 CDS probes related mainly to transport and binding, adaptive mechanisms, fatty acid biosynthesis, the cell membranes, bacteriophages, and transposon-related elements. The most prominent differences between the two clusters were related to resistance to toxic compounds frequently found in the environment, such as arsenic and cadmium, reflecting different adaptive responses in the evolution of the two clusters. Other relatively variable CDS groups were related to surface structures and the gram-positive cell wall, suggesting that the two clusters possess different antigenic properties. All the type B strains carried CDSs putatively related to capsule formation, which may play a role in adaptation to different environmental and clinical niches. Sequencing showed that representative strains of the two type B clusters both carried subtype B2 neurotoxin genes. As many of the type B strains studied have been isolated from foods or associated with botulism, it is expected that the two group I C. botulinum type B clusters present a public health hazard in Nordic countries. Knowing the genetic and physiological markers of these clusters will assist in targeting control measures against these pathogens.

Background: Retroviral LTRs, paired or single, influence the transcription of both retroviral and non-retroviral genomic sequences. Vertebrate genomes contain many thousand endogenous retroviruses (ERVs) and their LTRs. Single LTRs are difficult to detect from genomic sequences without recourse to repetitiveness or presence in a proviral structure. Understanding of LTR structure increases understanding of LTR function, and of functional genomics. Here we develop models of orthoretroviral LTRs useful for detection in genomes and for structural analysis. Principal Findings: Although mutated, ERV LTRs are more numerous and diverse than exogenous retroviral (XRV) LTRs. Hidden Markov models (HMMs), and alignments based on them, were created for HML- (human MMTV-like), general-beta-, gamma- and lentiretroviruslike LTRs, plus a general-vertebrate LTR model. Training sets were XRV LTRs and RepBase LTR consensuses. The HML HMM was most sensitive and detected 87% of the HML LTRs in human chromosome 19 at 96% specificity. By combining all HMMs with a low cutoff, for screening, 71% of all LTRs found by RepeatMasker in chromosome 19 were found. HMM consensus sequences had a conserved modular LTR structure. Target site duplications (TG-CA), TATA (occasionally absent), an AATAAA box and a T-rich region were prominent features. Most of the conservation was located in, or adjacent to, R and U5, with evidence for stem loops. Several of the long HML LTRs contained long ORFs inserted after the second A rich module. HMM consensus alignment allowed comparison of functional features like transcriptional start sites (sense and antisense) between XRVs and ERVs. Conclusion: The modular conserved and redundant orthoretroviral LTR structure with three A-rich regions is reminiscent of structurally relaxed Giardia promoters. The five HMMs provided a novel broad range, repeat-independent, ab initio LTR detection, with prospects for greater generalisation, and insight into LTR structure, which may aid development of LTR-targeted pharmaceuticals.

Perunaseitti on yksi tärkeimmistä taudeista, joka vähentää perunan satoa ja sadon laatua. Seittisieni (Rhizoctonia solani) säilyy siemenperunan pinnalla seittirupena ja maassa rihmastopahkoina, joista tartunta saa alkunsa. Taudin ensimmäinen vaihe ilmenee perunan idunkärkien kuolemisena, itujen haaroittumisena ja taimettumisen myöhästymisenä. Taimettuneiden varsien maanalaisiin osiin jää taudin näkyväksi merkiksi versolaikkuja. Miksi osa iduista pystyy kuitenkin tartunnasta huolimatta taimettumaan, on epäselvää. Tämän ilmiön tarkempi ymmärtäminen voisi edesauttaa perunaseitin haittavaikutusten lievittämistä kasvukauden alussa. Tutkimuksen tarkoituksena oli selvittää, aiheuttaako seittisienen tartunta puolustusreaktioita perunan iduissa. Mukulat idätettiin pimeässä olosuhteiltaan kontrolloidussa kasvatuskaapissa, jossa lämpötila vastasi taimettumisen kannalta suotuisia peltooloja. Olosuhteet suosivat myös seittisienen aiheuttaman versolaikun syntymistä. Koejärjestelyssä perunan idun alaosa tartutettiin seittisienellä. Idun yläosa eristettiin alaosasta muovikauluksella, jonka tarkoitus oli estää sienirihman kasvaminen erittäin infektioalttiiseen idun kärkeen. Perunan geenien ilmentymistä idun kärkiosassa tutkittiin mikrosiruanalyysillä 48 ja 120 tuntia alaosan tartutuksen jälkeen. Analyysissä voitiin havainnoida samanaikaisesti n. 10,000 geenin ilmentymistä (noin kolmannes perunan kaikista geeneistä). Tulokset osoittivat, että suuri määrä kasvien taudinkestävyyteen liittyviä geenejä aktivoitui itujen kärjissä, kun idun tyviosa tartutettiin seittisienellä. Nämä systeemiset puolustusvasteet olivat selvästi sienen aiheuttamia, sillä iduissa, joita ei tartutettu, samanlaista puolustuksen aktivoitumista ei tapahtunut. Rinnakkaisissa kokeissa tartutettiin idun kärkeä seittisienellä sen jälkeen, kun tyviosa oli tartutettu. Näissä iduissa kärjen havaittiin olevan huomattavasti kestävämpi seittitartunnalle kuin iduissa, joiden tyviosaa ei ollut ensin tartutettu seittisienellä. Kokeiden tulokset osoittivat, että perunan luontaiset puolustusreaktiot käynnistyvät seittisieni-infektion aikana, mikä selittänee, miksi osa iduista selviytyy seittitartunnasta ja pystyy taimettumaan. Näiden reaktioiden hyödynnettävyyttä seitintorjunnassa ei ole aiemmin tutkittu.

Groups I (proteolytic) and II (nonproteolytic) C. botulinum are genetically and physiologically distinct groups of organisms, with both groups being involved with human botulism. Due to differences in spore heat resistance and growth characteristics, the two groups possess different types of human health risks through foods, drink, and the environment. The epidemiology of human botulism due to Groups I and II C. botulinum is poorly understood, largely due to insufficient characterization of disease isolates, and warrants thorough outbreak investigation with a particular attention to discrimination between the different physiological groups of C. botulinum. In this study, a PCR assay was developed to discriminate between Group I and Group II C. botulinum. The assay is based on the fldB associated with phenylalanine metabolism in proteolytic clostridia, and employs an internal amplification control targeted to conservative regions of 16S rrn in Groups I and II C. botulinum. The assay correctly identified all 36 Group I and 24 Group II C. botulinum strains, possessing a 100% exclusivity and inclusivity. The assay provides a substantial improvement in discriminating between the Groups I and II C. botulinum, which traditionally is based on a time-consuming and error-prone culture method. Differentiation between the physiological groups of C. botulinum is an essential step in investigation of human botulism outbreaks, and should be considered as a diagnostic corner-stone in order to improve our epidemiological understanding of human botulism.

BackgroundDevelopment and maintenance of the identity of tissues is of central importance for multicellular organisms. Based on gene expression profiles, it is possible to divide genes in housekeeping genes and those whose expression is preferential in one or a few tissues and which provide specialized functions that have a strong effect on the physiology of the whole organism.ResultsWe have surveyed the gene expression in 78 normal human tissues integrating publicly available microarray gene expression data. A total amount of 1601 genes were identified as selectively expressed in one or more tissues. The tissue-selective genes covered a wide range of cellular and molecular functions, and could be linked to 361 human diseases with Mendelian inheritance. Based on the gene expression profiles, we were able to form a network of tissues reflecting their functional relatedness and, to certain extent, their development. Using co-citation driven gene network technique and promoter analysis, we predicted a transcriptional module where the co-operation of the transcription factors E2F and NF-kappaB can possibly regulate a number of genes involved in the neurogenesis that takes place in the adult hippocampus.ConclusionsHere we propose that integration of gene expression data from Affymetrix GeneChip experiments is possible through re-annotation and commonly used pre-processing methods. We suggest that some functional aspects of the tissues can be explained by the co-operation of multiple transcription factors that regulate the expression of selected groups of genes.

Genetic modification using gene transfer (GM) is still controversial when applied to plant breeding at least in Europe. One major concern is how GM affects other genes and thus the metabolism of the plant. In this study, 225 genetically modified lines of the ornamental plant Gerbera hybrida and 42 non-GM gerbera varieties were used to investigate changes in secondary metabolism. The cytotoxicity of GM and non-GM gerbera extracts was evaluated on human cell lines derived from lung, liver, and intestinal tissues. The results indicate that the safety profile for GM gerbera lines is similar to the viability pattern for non-GM varieties-none of the extracts were toxic. In addition, metabolic fingerprints of gerbera extracts were identified using thin-layer chromatography and analysed by principal component analysis (PCA), the nearest neighbour classifier, and Fligner-Killeen test. No new compounds unique to GM lines were observed. With PCA, no separation between GM gerbera lines and varieties could be demonstrated. In the nearest neighbour classifier, 54% of the samples found the expected neighbour based on the gene constructs used for transformation. With Fligner-Killeen test, we studied if the amounts of compounds vary more in GM gerberas than in varieties. In most cases, there were no statistically significant differences between the varieties and GM lines or there was more variation among the non-GM varieties than in the GM lines. The variance of a single compound was significantly larger in transgenic gerbera lines than in varieties and of three compounds in non-GM varieties.

The article is related to the development of techniques for automatic recognition of bird species by their sounds. It has been demonstrated earlier that a simple model of one time-varying sinusoid is very useful in classification and recognition of typical bird sounds. However, a large class of bird sounds are not pure sinusoids but have a clear harmonic spectrum structure. We introduce a way to classify bird syllables into four classes by their harmonic structure.

In this work an online algorithm is presented for the construction of the self-organizing map (SOM) of symbol strings. Each node of the SOM grid is associated with a model string which is a variable-vector sequence. Smooth interpolation method is applied in the training which performs simultaneous adaptation of the symbol content and the length of the model string. The efficiency of the method is demonstrated by the clustering of a 100,000-word English dictionary.

Time information of the input data is used for evaluating the goodness of the self-organizing map to store and represent temporal feature vector sequences. A new node neighborhood is defined for the map which takes the temporal order of the input samples into account. A connection is created between those two map modes which are the best-matching units for two successive input samples in time. This results in the time-topology preserving network.

This paper presents methods to improve the probability density estimation in hidden Markov models for phoneme recognition by exploiting the self-organizing map (SOM) algorithm. The advantage of using the SOM is based on the created approximative topology between the mixture densities by training the Gaussian mean vectors used as the kernel centers by the SOM algorithm. The topology allows the neighboring mixtures to respond strongly to the same inputs and so most of the nearest mixtures used to approximate the current observation probability will be found in the topological neighborhood of the "winner" mixture. Also the knowledge about the previous winners are used to speed up the search for the new winners. Tree-search SOMs and segmental SOM training are studied aiming at faster search and suitability for HMM training. The framework for the presented experiments includes mel-cepstrum features and phoneme-wise tied mixture density HMMs.

A method is presented for speeding up the performance of the HMM based speech recognition system where the states are modeled by a large number of Gaussian kernels. The emission probabilities of the states are usually dominated by the nearest Gaussians to the input vector. The speedup is gained without deteriorating the recognition accuracy by concentrating on these kernels in the reduced K-best-kernel search. In this work, the time information of the input is encoded to the connections of the kernels. The search for the dominating kernels is then performed along the kernel connections which model the trajectories of the speech in the feature space. In the experiments, speaker-dependent speech recognizers were trained for ten speakers. The number of distance computations between feature vectors and kernel mean vectors was reduced 75% without increasing the average phoneme recognition error, which was 5.7% for the baseline system.

The self-organizing map (SOM) represents an open set of input samples by a topologically organized, finite set of models. In this paper, a new version of the SOM is used for the clustering, organization, and visualization of a large database of symbol sequences (viz. protein sequences). This method combines two principles: the batch computing version of the SOM, and computation of the generalized median of symbol strings.

New, more effective software tools are needed for the analysis and organization of the continually growing biological databases. An extension of the Self-Organizing Map (SOM) is used in this work for the clustering of all the 77,977 protein sequences of the SWISS-PROT database, release 37. In this method, unlike in some previous ones, the data sequences are not converted into histogram vectors in order to perform the clustering. Instead, a collection of true representative model sequences that approximate the contents of the database in a compact way is found automatically, based on the concept of the generalized median of symbol strings, after the user has defined any proper similarity measure for the sequences such as Smith-Waterman, BLAST, or FASTA. The FASTA method is used in this work. The benefits of the SOM and also those of its extension are fast computation, approximate representation of the large database by means of a much smaller, fixed number of model sequences, and an easy interpretation of the clustering by means of visualization. The complete sequence database is mapped onto a two-dimensional graphic SOM display, and clusters of similar sequences are then found and made visible by indicating the degree of similarity of the adjacent model sequences by shades of gray.

To collect specific information about the genetic mechanisms that Clostridium botulinum strains utilise when adapting to changing environments, 16 C. botulinum genomes were analysed with comparative genome sequence analysis. Particular attention was paid to low temperature adaptation and the presence of cold shock protein coding genes in these genomes was evaluated. Surprisingly, unlike any other studied strains, the type E neurotoxin-producing strains lacked these extremely conserved genes. This finding suggests unique mechanisms for the cold tolerance of these strains and offers a new perspective into the investigations concerning this subject. The sizes of the pangenome and core genome of a certain bacterial species are considered to reflect the

Pectobacterium atrosepticum is a Gram-negative plant-pathogenic bacterium that rots potato stems and tubers. Microarray analysis was used to identify genes that were differentially expressed when host extracts were added to the growth medium. Potato extracts downregulated the expression of ribosomal genes and genes related to uptake and metabolism of nutrients, and upregulated genes needed for nitrate or phosphonate use. Some of the observed changes in gene expression in host-extract-induced cultures are similar to those during attachment of the bacterium to host tissues. Other responses indicated defence against toxic metabolites in the extract. Tuber extract induced a large gene cluster having homology to type VI secretion genes shown to be virulence determinants in many, but not all, animal and human pathogens. Two of the genes in the type VI cluster were found to be expressed during infection in potato tubers and stems, and mutants with knockouts of the corresponding genes had increased virulence on potato. One of the type VI secretion mutants was further characterized and found to grow to higher cell density in culture in the presence of host extract and to produce slightly more extracellular tissue-macerating enzymes than the wild-type strain. Analysis of secreted proteins showed that this type VI mutant was affected in the production of haemolysin-coregulated proteins (Hcps), which have been suggested to be secreted by the type VI pathway in other bacteria. The results suggest that the type VI secretion system of P. atrosepticum is needed for secretion of Hcps but not for virulence on its host plant, potato.