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In bioproduction processes cellular heterogeneity can cause unpredictable process outcomes or even provoke process failure. Still, cellular heterogeneity is not examined systematically in bioprocess research and development. One reason for this shortcoming are the applied average bulk analyses, which are not able to detect cell-to-cell differences. In this work we present a microfluidic tool for single-cell cultivation of mammalian suspension cells (MaSC). The design of our platform allows cultivation at highly controllable environments. As model system CHO K1 cells were cultivated over 150 h. Growth behavior was analyzed on single-cell level and resulted in growth rates between 0.85 - 1.16 d-1 , which are comparable to classical cultivation approaches such as shake flask and lab-scale bioreactor. At the same time, heterogeneous growth and division behavior, e.g., unequal division time, as well as rare cellular events like polynucleation or reversed mitosis were observed, which would have remained undetected in a standard population analysis based on average measurements. Therefore, MaSC will open the door for systematic single-cell analysis of mammalian suspension cells. Possible fields of application represent basic research topics like cell-to-cell heterogeneity studies, clonal stability, pharmaceutical drug screening and stem cell research, as well as bioprocess related topics such as media development and novel scale-down approaches. This article is protected by copyright. All rights reserved.

Actinoplanes sp. SE50/110 (ATCC 31044) is the wild type of industrial producer strains of acarbose. Acarbose has been used since the early 1990s as an inhibitor of intestinal human α-glucosidases in the medical treatment of type II diabetes mellitus. The small secreted protein Cgt, which consists of a single carbohydrate-binding module (CBM) 20-domain, was found to be highly expressed in Actinoplanes sp. SE50/110 in previous studies, but neither its function nor a possible role in the acarbose formation was explored, yet. Here, we demonstrated the starch-binding function of the Cgt protein in a binding assay. Transcription analysis showed that the cgt gene was strongly repressed in the presence of glucose or lactose. Due to this and its high abundance in the extracellular proteome of Actinoplanes, a functional role within the sugar metabolism or in the environmental stress protection was assumed. However, the gene deletion mutant ∆cgt, constructed by CRISPR/Cas9 technology, displayed no apparent phenotype in screening experiments testing for pH and osmolality stress, limited carbon source starch, and the excess of seven different sugars in liquid culture and further 97 carbon sources in the Omnilog Phenotypic Microarray System of Biolog. Therefore, a protective function as a surface protein or a function within the retainment and the utilization of carbon sources could not be experimentally validated. Remarkably, enhanced production of acarbose was determined yielding into 8–16% higher product titers when grown in maltose-containing medium.

Microalgae are rapidly growing, low-input requiring, sun light-utilizing microorganisms capable of converting carbon dioxide into various natural products, a major portion of which are hydrocarbons. Their cellular compartmentalization and photosynthetic apparatus depend on robust turnover of two hydrocarbon classes, isoprenoids and acyl-lipids. This review summarizes the current understanding of algal hydrocarbon metabolism, including carbon partitioning capacities, the localization and size of precursor pools, environmental effects on flux distribution, and limiting factors towards efficient (heterologous) hydrocarbon production. Questions and challenges regarding our knowledge of algal hydrocarbon metabolism as well as guidelines for systematic engineering are presented. Recent engineering achievements indicate fundamental plasticity in the (heterologous) hydrocarbon metabolism of green algae while highlighting their potential as renewable sources of these products. Copyright © 2019 Elsevier Ltd. All rights reserved.

Currently, little is known about the genetic background of restrictive cardiomyopathy (RCM). Herein, we screened an index patient with RCM in combination with atrial fibrillation using a next generation sequencing (NGS) approach and identified the heterozygous mutation DES-c.735G>C. As DES-c.735G>C affects the last base pair of exon-3, it is unknown whether putative missense or splice site mutations are caused. Therefore, we applied nanopore amplicon sequencing revealing the expression of a transcript without exon-3 in the explanted myocardial tissue of the index patient. Western blot analysis verified this finding at the protein level. In addition, we performed cell culture experiments revealing an abnormal cytoplasmic aggregation of the truncated desmin form (p.D214-E245del) but not of the missense variant (p.E245D). In conclusion, we show that DES-c.735G>C causes a splicing defect leading to exon-3 skipping of the DES gene. DES-c.735G>C can be classified as a pathogenic mutation associated with RCM and atrial fibrillation. In the future, this finding might have relevance for the genetic understanding of similar cases.

Corynebacterium glutamicum is a Gram-positive bacterium found in soil where the condition changes demand plasticity of the regulatory machinery. The study of such machinery at the global scale has been challenged by the lack of data integration. Here, we report three regulatory network models for C. glutamicum: strong (3040 interactions) constructed solely with regulations previously supported by directed experiments, all evidence (4665 interactions) containing the strong network, regulations previously supported by nondirected experiments, and protein–protein interactions with a direct effect on gene transcription, sRNA (5222 interactions) containing the all evidence network and sRNA-mediated regulations. Compared to the previous version (2018), the strong and all evidence networks increased by 75 and 1225 interactions, respectively. We analyzed the system-level components of the three networks to identify how they differ and compared their structures against those for the networks of more than 40 species. The inclusion of the sRNA-mediated regulations changed the proportions of the system-level components and increased the number of modules but decreased their size. The C. glutamicum regulatory structure contrasted with other bacterial regulatory networks. Finally, we used the strong networks of three model organisms to provide insights and future directions of the C.glutamicum regulatory network characterization.

The nematophagous fungus Hirsutella rhossiliensis is a common antagonist of a number of important plant-parasitic nematodes including the beet cyst nematode Heterodera schachtii. Although H. rhossiliensis is highly pathogenic, slow growth characteristics and weak competiveness under field conditions limit its biocontrol potential. Cellulose-based microcapsules were tested for their ability to improve fungal competitiveness in the soil and nematode parasitism. In a first approach microcapsules containing 15% fungal mycelium plus 15% corn gluten and 0.5% yeast extract as additives gave low levels of H. schachtii control. Fungal parasitism was not observed and indicated that nematode mortality was most likely caused by non-specific accumulation of nematicidal compounds associated with decomposition of the additives in the microcapsules. Removal of the corn gluten additive resulted in a minor increase of 20% parasitism of H. schachtii juveniles. Furthermore, the yeast extract that acted as nutrient source for the fungus diffused through the semi-permeable membrane of the capsules during the formulation process and was ineffective. Replacing the corn gluten and yeast extract with 3% autoclaved baker’s yeast and using finely dispersed mycelium instead of pelletized mycelium increased juvenile parasitism in the soil up to 90% and decreased number of juveniles per cm root length by over 80%. Cellulose-based microcapsules supplemented with autoclaved baker’s yeast, therefore, proved to be an effective delivery system for H. rhossiliensis to control H. schachtii.

BackgroundGrapevine cultivars of the Pinot family represent clonally propagated mutants with major phenotypic and physiological differences, such as different colour or shifted ripening time, as well as changes in important viticultural traits. Specifically, the cultivars ‘Pinot Noir’ (PN) and ‘Pinot Noir Precoce’ (PNP, early ripening) flower at the same time, but vary in the beginning of berry ripening (veraison) and, consequently, harvest time. In addition to genotype, seasonal climatic conditions (i.e. high temperatures) also affect ripening times. To reveal possible regulatory genes that affect the timing of veraison onset, we investigated differences in gene expression profiles between PN and PNP throughout berry development with a closely meshed time series and over two separate years.ResultsThe difference in the duration of berry formation between PN and PNP was quantified to be approximately two weeks under the growth conditions applied, using plant material with a proven PN and PNP clonal relationship. Clusters of co-expressed genes and differentially expressed genes (DEGs) were detected which reflect the shift in the timing of veraison onset. Functional annotation of these DEGs fit to observed phenotypic and physiological changes during berry development. In total, we observed 3,342 DEGs in 2014 and 2,745 DEGs in 2017 between PN and PNP, with 1,923 DEGs across both years. Among these, 388 DEGs were identified as veraison-specific and 12 were considered as berry ripening time regulatory candidates. The expression profiles revealed two candidate genes for ripening time control which we designatedVviRTIC1andVviRTIC2(VIT_210s0071g01145 and VIT_200s0366g00020, respectively). These genes likely contribute the phenotypic differences observed between PN and PNP.ConclusionsMany of the 1,923 DEGs show highly similar expression profiles in both cultivars if the patterns are aligned according to developmental stage. In our work, putative genes differentially expressed between PNP and PN which could control ripening time as well as veraison-specific genes were identified. We point out connections of these genes to molecular events during berry development and discuss potential candidate genes which may control ripening time. Two of these candidates were observed to be differentially expressed in the early berry development phase. Several down-regulated genes during berry ripening are annotated as auxin response factors / ARFs. Conceivably, general changes in auxin signaling may cause the earlier ripening phenotype of PNP.

Corynebacterium pseudotuberculosis is a Gram-positive, facultative intracellular, pathogenic bacterium that infects several different hosts, yielding serious economic losses in livestock farming. It causes several diseases including oedematous skin disease (OSD) in buffaloes, ulcerative lymphangitis (UL) in horses, and caseous lymphadenitis (CLA) in sheep, goats and humans. Despite its economic and medical-veterinary importance, our understanding concerning this organism’s transcriptional regulatory mechanisms is still limited. Here, we review the state of the art knowledge on transcriptional regulatory mechanisms of this pathogenic species, covering regulatory interactions mediated by two-component systems, transcription factors and sigma factors. Key transcriptional regulatory players involved in virulence and pathogenicity of C. pseudotuberculosis, such as the PhoPR system and DtxR, are in the focus of this review, as these regulators are promising targets for future vaccine design and drug development. We conclude that more experimental studies are needed to further understand the regulatory repertoire of this important zoonotic pathogen, and that regulators are promising targets for future vaccine design and drug development.

Controlled RNA degradation is a crucial process in bacterial cell biology for maintaining proper transcriptome homeostasis and adaptation to changing environments. mRNA turnover in many Gram-positive bacteria involves a specialized ribonuclease called RNase J (RnJ). To date, however, nothing is known about this process in the diphtheria-causative pathogen Corynebacterium diphtheriae, nor is known the identity of this ribonuclease in this organism. Here, we report that C. diphtheriae DIP1463 encodes a predicted RnJ homolog, comprised of a conserved N-terminal β-lactamase domain, followed by β-CASP and C-terminal domains. A recombinant protein encompassing the β-lactamase domain alone displays 5′-exoribonuclease activity, which is abolished by alanine-substitution of the conserved catalytic residues His186 and His188. Intriguingly, deletion of DIP1463/rnj in C. diphtheriae reduces bacterial growth and generates cell shape abnormality with markedly augmented cell width. Comparative RNA-seq analysis revealed that RnJ controls a large regulon encoding many factors predicted to be involved in biosynthesis, regulation, transport, and iron acquisition. One upregulated gene in the ∆rnj mutant is ftsH, coding for a membrane protease (FtsH) involved in cell division, whose overexpression in the wild-type strain also caused cell-width augmentation. Critically, the ∆rnj mutant is severely attenuated in virulence in a Caenorhabditis elegans model of infection, while the FtsH-overexpressing and toxin-less strains exhibit full virulence as the wild-type strain. Evidently, RNase J is a key ribonuclease in C. diphtheriae that post-transcriptionally influences the expression of numerous factors vital to corynebacterial cell physiology and virulence. Our findings have significant implications for basic biological processes and mechanisms of corynebacterial pathogenesis.

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Flavonol synthase (FLS) is a key enzyme for the formation of flavonols, which are a subclass of the flavonoids. FLS catalyses the conversion of dihydroflavonols to flavonols. The enzyme belongs to the 2-oxoglutarate-dependent dioxygenases (2-ODD) superfamily. We characterized the FLS gene family of Brassica napus that covers 13 genes, based on the genome sequence of the B. napus cultivar Express 617. The goal was to unravel which BnaFLS genes are relevant for seed flavonol accumulation in the amphidiploid species B. napus. Two BnaFLS1 homeologs were identified and shown to encode bifunctional enzymes. Both exhibit FLS activity as well as flavanone 3-hydroxylase (F3H) activity, which was demonstrated in vivo and in planta. BnaFLS1-1 and -2 are capable of converting flavanones into dihydroflavonols and further into flavonols. Analysis of spatio-temporal transcription patterns revealed similar expression profiles of BnaFLS1 genes. Both are mainly expressed in reproductive organs and co-expressed with the genes encoding early steps of flavonoid biosynthesis. Our results provide novel insights into flavonol biosynthesis in B. napus and contribute information for breeding targets with the aim to modify the flavonol content in rapeseed.

A growing body of evidence demonstrates the potential of various microbes to enhance plant productivity in cropping systems although their successful field application may be impaired by several biotic and abiotic constraints. In the present work, we aimed at developing multifunctional synthetic microbial consortia to be used in combination with suitable bioactive compounds for improving crop yield and quality. Plant growth-promoting microorganisms (PGPMs) with different functional attributes were identified by a bottom-up approach. A comprehensive literature survey on PGPMs associated with maize, wheat, potato and tomato, and on commercial formulations, was conducted by examining peer-reviewed scientific publications and results from relevant European projects. Metagenome fragment recruitments on genomes of potential PGPMs represented in databases were also performed to help identify plant growth-promoting (PGP) strains. Following evidence of their ability to coexist, isolated PGPMs were synthetically assembled into three different microbial consortia. Additionally, the effects of bioactive compounds on the growth of individually PGPMs were tested in starvation conditions. The different combination products based on microbial and non-microbial biostimulants (BS) appear worth considering for greenhouse and open field trials to select those potentially adoptable in sustainable agriculture.

Many bacteria encode so-called cold shock proteins. These proteins are characterized by a conserved protein domain. Often, the bacteria have multiple cold shock proteins that are expressed either constitutively or at low temperatures. In the Gram-positive model bacterium Bacillussubtilis, two of three cold shock proteins, CspB and CspD, belong to the most abundant proteins suggesting a very important function. To get insights into the role of these highly abundant proteins, we analyzed the phenotypes of single and double mutants, tested the expression of the csp genes and the impact of CspB and CspD on global gene expression in B. subtilis. We demonstrate that the simultaneous loss of both CspB and CspD results in a severe growth defect, in the loss of genetic competence, and the appearance of suppressor mutations. Overexpression of the third cold shock protein CspC could compensate for the loss of CspB and CspD. The transcriptome analysis revealed that the lack of CspB and CspD affects the expression of about 20% of all genes. In several cases, the lack of the cold shock proteins results in an increased read-through at transcription terminators suggesting that CspB and CspD might be involved in the control of transcription termination.

Cardiovascular diseases are the number one cause of morbidity and mortality worldwide, but the underlying molecular mechanisms remain not well understood. Cardiomyopathies are primary diseases of the heart muscle and contribute to high rates of heart failure and sudden cardiac deaths. Here, we distinguished four different genetic cardiomyopathies based on gene expression signatures. In this study, RNA-Sequencing was used to identify gene expression signatures in myocardial tissue of cardiomyopathy patients in comparison to non-failing human hearts. Therefore, expression differences between patients with specific affected genes, namely \(\it LMNA\) (lamin A/C), \(\it RBM20\) (RNA binding motif protein 20), \(\it TTN\) (titin) and \(\it PKP2\) (plakophilin 2) were investigated. We identified genotype-specific differences in regulated pathways, Gene Ontology (GO) terms as well as gene groups like secreted or regulatory proteins and potential candidate drug targets revealing specific molecular pathomechanisms for the four subtypes of genetic cardiomyopathies. Some regulated pathways are common between patients with mutations in \(\it RBM20\) and \(\it TTN\) as the splice factor RBM20 targets amongst other genes \(\it TTN\), leading to a similar response on pathway level, even though many differentially expressed genes (DEGs) still differ between both sample types. The myocardium of patients with mutations in \(\it LMNA\) is widely associated with upregulated genes/pathways involved in immune response, whereas mutations in \(\it PKP2\) lead to a downregulation of genes of the extracellular matrix. Our results contribute to further understanding of the underlying molecular pathomechanisms aiming for novel and better treatment of genetic cardiomyopathies.

Research in comparative genomics supports the investigation of important questions in molecular biology, genetics and biomedicine. A central question in this field is the elucidation of similarities and differences between genomes by means of different measures. This summary, submitted to CTD 2020, briefly describes the main contributions, originality and impact possibilities of the thesis entitled "Distance and Similarity Measures in Comparative Genomics", by Diego P. Rubert.

The structural modeling and representation of cells is a complex task as different microscopic, spectroscopic and other information resources have to be combined to achieve a three-dimensional representation with high accuracy. Moreover, to provide an appropriate spatial representation of the cell, a stereoscopic 3D (S3D) visualization is favorable. In this work, a structural cell model is created by combining information from various light microscopic and electron microscopic images as well as from publication-related data. At the mesoscopic level each cell component is presented with special structural and visual properties; at the molecular level a cell membrane composition and the underlying modeling method are discussed; and structural information is correlated with those at the functional level (represented by simplified energy-producing metabolic pathways). The organism used as an example is the unicellular Chlamydomonas reinhardtii, which might be important in future alternative energy production processes. Based on the 3D model, an educative S3D animation was created which was shown at conferences. The complete workflow was accomplished by using the open source 3D modeling software Blender. The discussed project including the animation is available from: http://Cm5.CELLmicrocosmos.org

Among green freshwater microalgae, Chlamydomonas reinhardtii has the most comprehensive and developed molecular toolkit, however, advanced genetic and metabolic engineering driven from the nuclear genome is generally hindered by inherently low transgene expression levels. Progressive strain development and synthetic promoters have improved the capacity of transgene expression; however, the responsible regulatory mechanisms are still not fully understood. Here, we elucidate the sequence specific dynamics of native regulatory element insertion into nuclear transgenes. Systematic insertions of the first intron of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit 2 (rbcS2i1) throughout codon-optimized coding sequences (CDS) generates optimized algal transgenes which express reliably in C. reinhardtii. The optimal rbcS2i1 insertion site for efficient splicing was systematically determined and improved gene expression rates were shown using a codon-optimized sesquiterpene synthase CDS. Sequential insertions of rbcS2i1 were found to have a step-wise additive effect on all levels of transgene expression, which is likely correlated to a synergy of transcriptional machinery recruitment and mimicking the short average exon lengths natively found in the C. reinhardtii genome. We further demonstrate the value of this optimization with five representative transgene examples and provide guidelines for the design of any desired sequence with this strategy.

Plant extracts from the neem tree can be applied as a bioinsecticidal compound in ‘attract-and-kill’ co-formulations to control soildwelling insect pests. Since insects are attracted to the bioinsecticide, the attract-and-kill approach benefits from a reduced dose of insecticide needed. Here, we demonstrate that a powdery neem seed kernel extract in combination with corn starch significantly enhances the drying survival of coencapsulated CO2-releasing Saccharomyces cerevisiae. Additionally, an increased content of the neem extract was shown to slow down the relative release of azadirachtin A. Both findings are of high relevance for the development of formulations in the field of biological pest control.

Biocontrol of insect pests using fungal entomopathogens has been promoted as a promising alternative to chemical pesticides. Apart from their direct mode of action against insect pests, several fungal entomopathogens have been reported as natural endophytes, paving the way for novel plant protection measures. In this study, we aimed at the selective propagation of finely dispersed Metarhizium brunneum strain BIPESCO5 mycelium in submerged culture and encapsulation in calcium alginate/starch beads to protect the fungus during drying, enable growth on different soils and promote endophytism in tomato plants. We found that a combination of osmotic pressure and increased medium viscosity promoted selective formation of finely dispersed mycelium reflected by 34.1-fold decreased pellet diameters, 1.8-fold increased mycelial biomass concentrations and low blastospore contents of 40.4 × 105 mL−1 after 48 h. Encapsulation of mycelium enhanced drying survival by 31.5%. Co-encapsulated starch was degraded by 24.8% after 21 days and served as a nutrient source for growth on soils with best results on sterile and non-sterile potting substrate with 8.7 mm and 4.4 mm radial mycelial growth, respectively, compared to water agar with 7.6 mm. When applied to the soil, encapsulation significantly increased endophytism 3.8–7.0-fold compared to plants treated with non-formulated fungal biomass. This study provides the first evidence of endophytic establishment of M. brunneum in the stem of tomato plants after application of mycelium to roots as well as on increased endophytism by encapsulation. These results might provide the basis for future work on increasing endophytism by formulation technologies.

The construction of microbial platform organisms by means of genome reduction is an ongoing topic in biotechnology. In this study, we investigated whether the deletion of single or multiple gene clusters has a positive effect on the secretion of cutinase from Fusarium solani pisi in the industrial workhorse Corynebacterium glutamicum. A total of 22 genome‐reduced strain variants were compared applying two Sec signal peptides from Bacillus subtilis. High‐throughput phenotyping using robotics‐integrated microbioreactor technology with automated harvesting revealed distinct cutinase secretion performance for a specific combination of signal peptide and genomic deletions. Strikingly, bioreactor fed‐batch cultivations at controlled growth rates resulted in a complete reversal of the batch screening results, with the cutinase yield for strain GRS41_51_NprE dropping by ~ 25% compared to the reference strain. Thus, the choice of bioprocess conditions may turn a “high‐performance” strain from batch screening into a “low‐performance” strain in fed‐batch cultivation., Summary The construction of microbial platform organisms by means of genome reduction is an ongoing topic in biotechnology. In this study, we investigated whether the deletion of single or multiple gene clusters has a positive effect on the secretion of cutinase from Fusarium solani pisi in the industrial workhorse Corynebacterium glutamicum. A total of 22 genome‐reduced strain variants were compared applying two Sec signal peptides from Bacillus subtilis. High‐throughput phenotyping using robotics‐integrated microbioreactor technology with automated harvesting revealed distinct cutinase secretion performance for a specific combination of signal peptide and genomic deletions. The biomass‐specific cutinase yield for strain GRS41_51_NprE was increased by ~ 200%, although the growth rate was reduced by ~ 60%. Importantly, the causative deletions of genomic clusters cg2801‐cg2828 and rrnC‐cg3298 could not have been inferred a priori. Strikingly, bioreactor fed‐batch cultivations at controlled growth rates resulted in a complete reversal of the screening results, with the cutinase yield for strain GRS41_51_NprE dropping by ~ 25% compared to the reference strain. Thus, the choice of bioprocess conditions may turn a ‘high‐performance’ strain from batch screening into a ‘low‐performance’ strain in fed‐batch cultivation. In conclusion, future studies are needed in order to understand metabolic adaptations of C. glutamicum to both genomic deletions and different bioprocess conditions.

Mycobacterium avium (M. a.) subsp. paratuberculosis (MAP) is a worldwide-distributed obligate pathogen in ruminants causing Johne&rsquo, s disease. Due to a lack of complete subtype III genome sequences, there is not yet conclusive information about genetic differences between strains of cattle (MAP-C, type II) and sheep (MAP-S) type, and especially between MAP-S subtypes I, and III. Here we present the complete, circular genome of MAP-S/type III strain JIII-386 (DE) closed by Nanopore-technology and its comparison with MAP-S/type I closed genome of strain Telford (AUS), MAP-S/type III draft genome of strain S397 (U.S.), twelve closed MAP-C strains, and eight closed M.-a.-complex-strains. Structural comparative alignments revealed clearly the mosaic nature of MAP, emphasized differences between the subtypes and the higher diversity of MAP-S genomes. The comparison of various genomic elements including transposases and genomic islands provide new insights in MAP genomics. MAP type specific phenotypic features may be attributed to genes of known large sequence polymorphisms (LSPSs) regions I&ndash, IV and deletions #1 and #2, confirmed here, but could also result from identified frameshifts or interruptions of various virulence-associated genes (e.g., mbtC in MAP-S). Comprehensive core and pan genome analysis uncovered unique genes (e.g., cytochromes) and genes probably acquired by horizontal gene transfer in different MAP-types and subtypes, but also emphasized the highly conserved and close relationship, and the complex evolution of M.-a.-strains.

Successful commercialization of microbial biocontrol agents, such as Metarhizium spp., is often constrained by poor drying survival and shelf life. Here, we hypothesized that culture age would influence endogenous arabitol, erythritol, mannitol and trehalose contents in M. brunneum mycelium and that elevated levels of these compounds would improve drying survival and shelf life of encapsulated mycelium coupled with enhanced fungal virulence against T. molitor larvae. We found that culture age significantly influenced endogenous arabitol and mannitol contents in mycelium with highest concentrations of 0.6 +/- 0.2 and 2.1 +/- 0.2 A mu g/mg after 72 h, respectively. Drying survival of encapsulated mycelium was independent of culture age and polyol content with 41.1 +/- 4.4 to 55.0 +/- 6.2%. Best shelf life was determined for biomass harvested after 72 h at all investigated storage temperatures with maximum values of 59.5 +/- 3.3% at 5 A degrees C followed by 54.5 +/- 1.6% at 18 A degrees C and 19.4 +/- 1.3% at 25 A degrees C after 6 months. Finally, high fungal virulence against T. molitor larvae of 83.3 +/- 7.6 to 98.0 +/- 1.8% was maintained during storage of encapsulated mycelium for 12 months with larval mortalities being independent of culture age and polyol content. In conclusion, our findings indicate beneficial effects of endogenous polyols in improving shelf life of encapsulated mycelium and this may spur the successful development of microbial biocontrol agents in the future.

Photosynthetic microalgae harbor enormous potential as light-driven green-cell factories for sustainable bio-production of a range of natural and heterologous products such as isoprenoids. Their capacity for photosynthesis and rapid low-input growth with (sun)light and CO2 is coupled to a robust metabolic architecture structured toward the generation of isoprenoid pigments and compounds involved in light capture, electron transfer, and radical scavenging. Metabolic engineering approaches using eukaryotic green microalgae have previously been hampered mainly by low-levels of nuclear transgene expression. Here, we employed a strategy of optimized transgene design which couples codon optimization and synthetic intron spreading for the expression of heterologous plant enzymes from the algal nuclear genome. The diterpenoids casbene, taxadiene, and 13R(+) manoyl oxide were produced after expressing heterologous diterpene synthases and enzymes participating in the 2- C -methyl- d -erythritol 4-phosphate (MEP) pathway which were all targeted to the algal chloroplast. Additionally, a truncated and soluble plant microsomal cytochrome P450 monooxygenase was functionally expressed and able to hydroxylate 13R(+) manoyl oxide when directed into the chloroplasts. The heterologous diterpenoids were found to be excreted from the cells and accumulate in dodecane solvent-culture overlays. It was shown that the algal cell could tolerate significant metabolic pull towards diterpenoids without loss of native pigments. Using an algal strain producing 13R(+) manoyl oxide as a model, diterpenoid production was shown to be highest in photoautotrophic cultivations using CO2 as the sole carbon source and day:night illumination cycles. Up to 80 mg 13R(+) manoyl oxide per gram cell dry mass ( CDM ) could be produced from C. reinhardtii in a 7 day batch cultivation with a sustained maximal productivity of 22.5 mg g cdm −1 d−1 over 3 consecutive days. Collectively the results presented here suggest that green algal cells have remarkable potential for the heterologous production of non-native isoprenoids and support the use of these hosts for (sun)light driven bioproduction concepts.

Background and aims Recent studies indicate the potential of endophytic entomopathogenic fungi to promote plant growth but little is known about the responses of root architecture to fungal endophytism. This study investigates potential adaptations of root architecture upon Metarhizium brunneum endophytism linked to improved plant growth and nutrition. Methods Plants (Solanum tuberosum L.) were grown in the presence of M. brunneum applied either as nonformulated mycelium or as mycelium containing beads. After 35 days, fungal growth, root endophytism, plant biomass and nutrition as well as root attributes were determined. Results In response to endophytism promoted by bead application, plant P contents and biomass were significantly increased, while N contents and shoot allocation were also significantly increased in plants from the beads without mycelium group. Bead application resulted in a shift from fine to medium-sized roots and in an increase in the number of root forks, while root diameter, surface area and the number of root tips and crossings were independent of either bead or M. brunneum treatment. Conclusions M. brunneum containing beads supported endophytism allowing for increases in plant P contents and biomass. However, root architecture was not strongly modulated by M. brunneum endophytism with N provision through bead application being more important than fungal P delivery.

There is growing evidence that entomopathogenic fungi play a role in plant growth promotion as colonizers of internal plant tissues. However, little is known about their potential to mitigate plant abiotic stress. Here, we investigated the influence of soil fertility on Metarhizium brunneum strain CB15 endophytism levels in potato plants linked to plant productivity and vitality. By application of encapsulated M. brunneum, the endophytism level, i.e. % presence of fungus in plant tissue, increased significantly under nutrient poor conditions. This correlated with significantly improved quantum yields of photosystem II. In addition, water use efficiency was increased with higher levels of M. brunneum endophytism. Furthermore, biomass, leaf area, nitrogen and phosphorus contents were enhanced indicating fungal nutrient mobilization and transfer. Our results provide first evidence for the role of entomopathogenic fungi in mitigating nutrient deficits in soil by improving plant productivity and vitality which may increase their use in plant protection strategies.

Highlights • P. saccharofermentans str. M3/6T harbors several carbohydrate active enzymes. • The strain encodes 64 polysaccharide utilization loci (PULs). • Several PULs are associated with genes encoding carbohydrate active enzymes. • P. saccharofermentans M3/6T harbors two CRISPR-cas systems., Proteiniphilum saccharofermentans str. M3/6T is a recently described species within the family Porphyromonadaceae (phylum Bacteroidetes), which was isolated from a mesophilic laboratory-scale biogas reactor. The genome of the strain was completely sequenced and manually annotated to reconstruct its metabolic potential regarding biomass degradation and fermentation pathways. The P. saccharofermentans str. M3/6T genome consists of a 4,414,963 bp chromosome featuring an average GC-content of 43.63%. Genome analyses revealed that the strain possesses 3396 protein-coding sequences. Among them are 158 genes assigned to the carbohydrate-active-enzyme families as defined by the CAZy database, including 116 genes encoding glycosyl hydrolases (GHs) involved in pectin, arabinogalactan, hemicellulose (arabinan, xylan, mannan, β-glucans), starch, fructan and chitin degradation. The strain also features several transporter genes, some of which are located in polysaccharide utilization loci (PUL). PUL gene products are involved in glycan binding, transport and utilization at the cell surface. In the genome of strain M3/6T, 64 PUL are present and most of them in association with genes encoding carbohydrate-active enzymes. Accordingly, the strain was predicted to metabolize several sugars yielding carbon dioxide, hydrogen, acetate, formate, propionate and isovalerate as end-products of the fermentation process. Moreover, P. saccharofermentans str. M3/6T encodes extracellular and intracellular proteases and transporters predicted to be involved in protein and oligopeptide degradation. Comparative analyses between P. saccharofermentans str. M3/6T and its closest described relative P. acetatigenes str. DSM 18083T indicate that both strains share a similar metabolism regarding decomposition of complex carbohydrates and fermentation of sugars.

Encapsulation of hydrolases in biopolymer-based hydrogels often suffers from low activities and encapsulation efficiencies along with high leaching and unsatisfactory recycling properties. Exemplified for the encapsulation of pig liver esterase the coating of alginate and chitosan beads have been studied by creating various biopolymer hydrogel beads. Enzyme activity and encapsulation efficiency were notably enhanced by chitosan coating of alginate beads while leaching remained nearly unchanged. This was caused by the enzymatic reaction acidifying the matrix, which increased enzyme retention through enhanced electrostatic enzyme-alginate interaction but decreased activity through enzyme deactivation. A practical and ready-to-use method for visualising pH in beads during reaction by co-encapsulation of a conventional pH indicator was also found. Our method proves that pH control inside the beads can only be realised by buffering. The resulting beads provided a specific activity of 0.267 μmol ∙ min-1 ∙ mg-1, effectiveness factor 0.88, encapsulation efficiency of 88%, 5% leaching and good recycling properties. This work will contribute towards better understanding and application of encapsulated hydrolases for enzymatic syntheses.

Since several years it is known that strains of the entomopathogenic fungus Beauveria bassiana (Balsamo-Crivelli) Vuillemin (Ascomycota: Hypocreales) are able to colonize plants as a true endophyte. However, so far no integrated bioprocess engineering approaches have been published where fermentation and formulation strategies are combined to optimize colonization of oilseed rape plant tissues. We therefore aimed at investigating whether and how blastospore (BS) formation can be shifted to resilient submerged conidiospores (SCS) by introducing osmotic stress in different growth phases. When 50 g/L NaCl was added after 48 h to a culture of B. bassiana a yield of 1.4 ± 0.1 × 10 10 SCS/g sucrose in shake flasks and 1.8 ± 0.1 × 10 10 SCS/g sucrose in a stirred tank reactor were obtained. In a bioreactor, 24 h after the addition of NaCl, the formation of BS slowed down, the respiratory quotient decreased and a shift from BS to SCS set in. Following these steps, different formulation strategies, namely encapsulation, film coating and liquid formulation were evaluated. B. bassiana grew out of beads as well as on commercial fungicide-coated seeds. Due to the complete suppression of fungal growth on non-sterile soil, the most suitable option was a foliar application. A liquid formulation consisting of 0.1% Triton X-114, 1% molasses, 1% titanium dioxide and 10 6 spores/mL was applied on leaf tips. After 14 days, the endophyte was detected by PCR and microscopic analysis in the leaves. Further research should focus on formation of SCS and protection of plants colonized by B. bassiana against herbivorous insects.

The recent discovery that entomopathogenic fungi can grow endophytically in plant tissues has spurred research into novel plant protection measures. However, current applications of fungi aiming at endophytism mostly lack targeted formulation strategies resulting in low efficacy. Here, we aimed at enhancing Metarhizium brunneum CB15 endophytism in potato plants by (i) improvement of fungal growth from beads and (ii) cellulase formation or addition to encapsulated mycelium. We found that beads supplemented with cellulose alone or in addition with inactivated baker's yeast exhibited cellulase activity and increased mycelial growth by 12.6 % and 13.6 %, respectively. Higher enzymatic activity achieved by cellulase co-encapsulation promoted a shift from mycelial growth to spore formation with maximum numbers of 2.5 × 108 ± 6.1 × 107 per bead. This correlated with improved endophytism in potato plants by 61.2 % compared to non-supplemented beads. Our study provides first evidence that customized formulations of fungal entomopathogens with enzymes can improve endophytism and this may increase efficacy in plant protection strategies against herbivorous pests.

Advanced biotechnology makes it possible to access a multitude of heterogeneous proteomic, interactomic, genomic, and functional annotation data. One challenge in computational biology is to integrate these data to enable automated prediction of the Subcellular Localizations (SCL) of human proteins. For proteins that have multiple biological roles, their correct in silico assignment to different SCL can be considered as an imbalanced multi-label classification problem. In this study, we developed a Bayesian Collective Markov Random Fields (BCMRFs) model for multi-SCL prediction of human proteins. Given a set of unknown proteins and their corresponding protein-protein interaction (PPI) network, the SCLs of each protein can be inferred by the SCLs of its interacting partners. To do so, we integrate PPIs, the adjacency of SCLs and protein features, and perform transductive learning on the re-balanced dataset. Our experimental results show that the spatial adjacency of the SCLs improves multi-SCL prediction, especially for the SCLs with few annotated instances. Our approach outperforms the state-of-art PPI-based and feature-based multi-SCL prediction method for human proteins.

CO2 is known as a major attractant for many arthropod pests which can be exploited for pest control within novel attract-and-kill strategies. This study reports on the development of a slow-release system for CO2 based on calcium alginate beads containing granular corn starch, amyloglucosidase and Saccharomyces cerevisiae. Our aim was to evaluate the conditions which influence the CO2 release and to clarify the biochemical reactions taking place within the beads. The amyloglucosidase was immobilized with a high encapsulation efficiency of 87% in Ca-alginate beads supplemented with corn starch and S. cerevisiae biomass. The CO2 release from the beads was shown to be significantly affected by the concentration of amyloglucosidase and corn starch within the beads as well as by the incubation temperature. Beads prepared with 0.1 amyloglucosidase units/g matrix solution led to a long-lasting CO2 emission at temperatures between 6 and 25 °C. Starch degradation data correlated well with the CO2 release from beads during incubation and scanning electron microscopy micrographs visualized the degradation of corn starch granules by the co-encapsulated amyloglucosidase. By implementing MALDI-ToF mass spectrometry imaging for the analysis of Ca-alginate beads, we verified that the encapsulated amyloglucosidase converts starch into glucose which is immediately consumed by S. cerevisiae cells. When applied into the soil, the beads increased the CO2 concentration in soil significantly. Finally, we demonstrated that dried beads showed a CO2 production in soil comparable to the moist beads. The long-lasting CO2- releasing beads will pave the way towards novel attract-and-kill strategies in pest control.

Calcium chloride (CC) is the most common cross-linker for the encapsulation of biocontrol microorganisms in alginate beads. The aim of this study was to evaluate if calcium gluconate (CG) can replace CC as cross-linker and at the same time improve viability after drying and rehydration, hygroscopic properties, shelf life and nutrient supply. Hence, the biocontrol fungi Metarhizium brunneum and Saccharomyces cerevisiae were encapsulated in Ca-alginate beads supplemented with starch. Beads were dried and maximum survival was found in beads cross-linked with CG. Beads prepared with CG showed lower hygroscopic properties, but a higher shelf life for encapsulated fungi. Moreover, we demonstrated that gluconate has a nutritive effect on encapsulated fungi, leading to increased mycelium growth of M. brunneum and to enhanced CO2 release from beads containing Saccharomyces cerevisiae. The application of CG as cross-linker will pave the way towards increasing drying survival and shelf life of various, especially drying-sensitive microbes.

Wireworms (Coleoptera: Elateridae) have recently become an increasing problem as agricultural insect pests due to the phasing out of effective control options. Entomopathogenic fungi such as Metarhizium brunneum have proven to be a promising microbial antagonist for wireworm control. Here, we tested whether the efficacy of M. brunneum can be increased through a combination with CO2, emitted by Saccharomyces cerevisiae, as an attractant (=attract-and-kill). We aimed at a technical scale production of a formulated biological control agent offering a practical and economically feasible application for wireworm control. Therefore, a novel technical formulation process for encapsulated S. cerevisiae (Attract beads) and M. brunneum (Kill beads) was investigated. For the bead production by jet cutting, the parameters nozzle diameter, pump speed, cutting device speed and collecting distance were evaluated. In order to dry the beads in a short time while maintaining a high cell viability, different drying temperatures during fluidised-bed drying were tested and the best results were obtained with an inlet air temperature profile between 50°C and 40°C. CO2 production of the beads in the soil was highest for co-applied Attract and Kill beads. The potential of beads to modify wireworm behaviour (Agriotes sputator) was tested in a rhizotron experiment. The Attract-and-Kill treatment (co-applied beads) significantly attracted wireworms, whereas Attract beads and Kill beads alone showed a weak, but non-significant attraction. Wireworm mortality could not be enhanced due to a low rate of mycosis from M. brunneum infection.

A Lolium perenne ice-binding protein (LpIBP) demonstrates superior ice recrystallization inhibition (IRI) activity and has proposed applications in cryopreservation, food texturing, as well as in being a "green" gas hydrate inhibitor. Recombinant production of LpIBP has been previously conducted in bacterial and yeast systems for studies of protein characterization, but large-scale applications have been hitherto limited due to high production costs. In this work, a codon-optimized LpIBP was recombinantly expressed and secreted in a novel one-step vector system from the nuclear genome of the green microalga Chlamydomonas reinhardtii. Both mixotrophic and photoautotrophic growth regimes supported LpIBP expression, indicating the feasibility of low-cost production using minimal medium, carbon dioxide, and light energy as input. In addition, multiple growth and bioproduct extraction cycles were performed by repetitive batch cultivation trials, demonstrating the potential for semi-continuous production and biomass harvesting. Concentrations of recombinant protein reached in this proof of concept approach were sufficient to demonstrate IRI activity in culture media without additional purification or concentration, with activity further verified by thermal hysteresis and morphology assays. The incorporation of the recombinant LpIBP into a model gas hydrate offers the promise that algal production may eventually find application as a "green" hydrate inhibitor.

Western corn rootworm larvae are serious soil dwelling maize pests, and use carbon dioxide (CO2) to locate maize roots. The efficacy of insecticides can be enhanced by a combination with an attractant used in host finding, known as attract and kill. This study tested the use of CO2 emitting capsules as an attractant in combination with the soil insecticide tefluthrin. An observation device was developed to study the temporal and spatial distribution changes of the larvae and to test whether these are influenced by the application of the capsules. Furthermore it was evaluated to what extent larvae are killed by the insecticide in combination with the capsules and whether this could be used for an attract and kill strategy to manage this pest. The observation device enabled recovery of 20–40% of the inserted larvae. The spatial analysis of distance indices revealed a sequence of spatial and temporal distribution patterns of the larvae in the root system. This sequence of spatial distribution was disrupted by an application of the capsules around which the larvae started to aggregate. Up to 40% mortality of the larvae with attract and kill was observed and thus could be increased over the conventional application (11% mortality) at lower application rates of tefluthrin. In conclusion an attract and kill strategy might be valuable to target this soil dwelling pest. Experiments under field conditions are needed to explore its potential as a management option for the western corn rootworm. Moreover, a further development of the capsules with host specific cues is needed to increase the attractiveness and subsequent mortality of the larvae.

Innovative wireworm control strategies are required to implement integrated pest management on the basis of the (EC) No regulation 1107/2009 and Directive 2009/128/EC. Entomopathogenic fungi, such as Metarhizium brunneum (Metschnikoff) Sorokin, are potential biological control agents for wireworm control but do not achieve high control efficacies in the field when applied as a conidia suspension. In a 2-year study, wireworm control with a novel attract-and-kill strategy aimed at enhancing M. brunneum efficacies in organic potato production systems in Lower Saxony, Germany. The approach is based on the attraction of wireworms (Agriotes spp. Eschscholtz) towards an artificial carbon dioxide-emitting source, using baker’s yeast (Saccharomyces cerevisiae Meyen ex Hansen) in combination with M. brunneum conidia for wireworm infection. Both components were encapsulated in alginate as a carrier material and applied in a mixture with two types of beads (one for encapsulated yeast and one for M.brunneum conidia). An application of these beads within the potato rows during potato planting reduced wireworm tuber damage by 37–75% relative to the untreated control and was able to enhance the efficacy of M. brunneum by up to 35% through an attract-and-kill approach compared to beads without a carbon dioxide source only. This strategy offers a high potential to promote biological wireworm control as an alternative to insecticide use by potentially reducing the inoculum compared to an inundate M. brunneum conidia release strategy.

T-DNA insertion mutants are very valuable for reverse genetics in Arabidopsis thaliana. Several projects have generated large sequence-indexed collections of T-DNA insertion lines, of which GABI-Kat is the second largest resource worldwide. User access to the collection and its Flanking Sequence Tags (FSTs) is provided by the front end SimpleSearch (http://www.GABI-Kat.de). Several significant improvements have been implemented recently. The database now relies on the TAIRv10 genome sequence and annotation dataset. All FSTs have been newly mapped using an optimized procedure that leads to improved accuracy of insertion site predictions. A fraction of the collection with weak FST yield was re-analysed by generating new FSTs. Along with newly found predictions for older sequences about 20 000 new FSTs were included in the database. Information about groups of FSTs pointing to the same insertion site that is found in several lines but is real only in a single line are included, and many problematic FST-to-line links have been corrected using new wet-lab data. SimpleSearch currently contains data from � 71 000 lines with predicted insertions covering 62.5% of the 27 206 nuclear protein coding genes, and offers insertion allele-specific data from 9545 confirmed lines that are available from the Nottingham Arabidopsis Stock Centre.

Firing of an individual neuron is determined by the activity of its presynaptic input ensemble. In this study we analyzed how presynaptic signals with different dynamics interact to control postsynaptic activity. In the blowfly's visual system we simultaneously recorded in vivo from an identified motion-sensitive neuron and from elements of the presynaptic ensemble. The presynaptic cells themselves are mutually electrically coupled and convey both graded and spike signals to their common postsynaptic target. We elicited spikes in the postsynaptic neuron by voltage-clamping one of the presynaptic neurons to various holding potentials and then analyzed the time course of the holding current. Current transients in the clamped presynaptic cell were found to coincide with postsynaptic spikes. The current transients were highly variable in amplitude and occasionally absent during postsynaptic spiking. These characteristics indicate that the current transients in the voltage-clamped neuron result from spikes in electrically coupled co-members of the presynaptic ensemble. Our results suggest that electrical coupling among presynaptic neurons mediates synchronization of spikes within the cell ensemble. Moreover, our findings demonstrate that the graded response component of the presynaptic cells effectively controls the postsynaptic firing rate on a coarse scale while the precise timing of the postsynaptic spikes is a consequence of spikes superimposed on the graded signals of the presynaptic neurons.

Pseudomonas aeruginosa is a major nosocomial bacterial pathogen causing complicated catheter-associated urinary tract infections (CAUTIs). Here, we present the 6.9-Mb draft genome sequence of P. aeruginosa MH38 isolated from an acute nosocomial CAUTI. It exhibits resistance to several antibiotics but revealed low-level production of virulence factors.

The larvae of the invasive maize pest Diabrotica virgifera virgifera (Coleoptera; Chrysomelidae, western corn rootworm) hatch in the soil in spring and search for maize roots following CO2 gradients. CO2 is one cue that might be used as an attractant towards soil insecticides, a mechanism already shown in laboratory experiments. This study compared the efficacy of several combinations of in or between-row applications of different rates of CO2-emitting capsules and/or soil insecticides (here tefluthrin) aimed at preventing root damage by the pest larvae under field conditions. CO2 emission of the capsules in the soil lasted up to 28 days with a peak after 21 days coinciding with the first larval hatch. The pest density in the soil was not high enough to cause root damage above the economic threshold. Furthermore all tefluthrin applications, regardless of whether at full, half or quarter rates effectively prevented root damage; thus CO2 did not significantly further increase this efficacy. In-row applications of tefluthrin with or without CO2-emitting capsules prevented root damage to a much larger extent (59–77 % on the node injury scale) than the between-row applications of tefluthrin with or without capsules (17–31 %). In conclusion, further research on belowground orientation and movement of D. v. virgifera larvae, as well as tests with combinations of CO2-emitting capsules and lower rates of soil insecticide are needed to potentially develop attract and kill strategies as a management option against this maize pest.

Because of the rising demand for microbial biological control agents, research into novel formulation methods, especially bioencapsulation, has notably increased in the past years. The aim of this review is to present a detailed illustrated overview on current encapsulation methods that are applied or that may be tailored to living biological control agents, especially microbial organisms and entomopathogenic nematodes. Capsules are manufactured by forming droplets from liquids and solidifying the liquid droplets to form particles. In this review, the methods are presented according to the manner of droplet formation (dripping and emulsification) and are subsequently categorized by the process of gelation or membrane formation. In a further category this review expands on coating methods using polyelectrolytes with altering charges. We put emphasis on chemical aspects which seem especially useful for scientists working in biological control.

Background Modern analytical methods in biology and chemistry use separation techniques coupled to sensitive detectors, such as gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). These hyphenated methods provide high-dimensional data. Comparing such data manually to find corresponding signals is a laborious task, as each experiment usually consists of thousands of individual scans, each containing hundreds or even thousands of distinct signals. In order to allow for successful identification of metabolites or proteins within such data, especially in the context of metabolomics and proteomics, an accurate alignment and matching of corresponding features between two or more experiments is required. Such a matching algorithm should capture fluctuations in the chromatographic system which lead to non-linear distortions on the time axis, as well as systematic changes in recorded intensities. Many different algorithms for the retention time alignment of GC-MS and LC-MS data have been proposed and published, but all of them focus either on aligning previously extracted peak features or on aligning and comparing the complete raw data containing all available features. Results In this paper we introduce two algorithms for retention time alignment of multiple GC-MS datasets: multiple alignment by bidirectional best hits peak assignment and cluster extension (BIPACE) and center-star multiple alignment by pairwise partitioned dynamic time warping (CeMAPP-DTW). We show how the similarity-based peak group matching method BIPACE may be used for multiple alignment calculation individually and how it can be used as a preprocessing step for the pairwise alignments performed by CeMAPP-DTW. We evaluate the algorithms individually and in combination on a previously published small GC-MS dataset studying the Leishmania parasite and on a larger GC-MS dataset studying grains of wheat (Triticum aestivum). Conclusions We have shown that BIPACE achieves very high precision and recall and a very low number of false positive peak assignments on both evaluation datasets. CeMAPP-DTW finds a high number of true positives when executed on its own, but achieves even better results when BIPACE is used to constrain its search space. The source code of both algorithms is included in the OpenSource software framework Maltcms, which is available from http://maltcms.sf.net. The evaluation scripts of the present study are available from the same source.

The brain combines and integrates multiple cues to take coherent, context-dependent action using distributed, event-based computational primitives. Computational models that use these principles in software simulations of recurrently coupled spiking neural networks have been demonstrated in the past, but their implementation in hybrid analog/ digital Very Large Scale Integration (VLSI) spiking neural networks remains challenging. Here, we demonstrate a distributed spiking neural network architecture comprising multiple neuromorphic VLSI chips able to reproduce these types of cue combination and integration operations. This is achieved by encoding cues as population activities of input nodes in a network of recurrently coupled VLSI Integrate-and-Fire (I&F) neurons. The value of the cue is place-encoded, while its uncertainty is represented by the width of the population activity profile. Relationships among different cues are specified through bidirectional connectivity matrices, shared between the individual input node populations and an intermediate node population. The resulting network dynamics bidirectionally relate not only the values of three variables according to a specified relation, but also their uncertainties. When cues on two populations are specified, the standard deviation of the activity in the unspecified population varies approximately linearly with the widths of the two input cues, and has less than 6% error in position compared to the value specified by the inputs. The results suggest a mechanism for recurrently relating cues such that missing information can both be recovered and assigned a level of certainty.

Axonal delays are used in neural computation to implement faithful models of biological neural systems, and in spiking neural networks models to solve computationally demanding tasks. While there is an increasing number of software simulations of spiking neural networks that make use of axonal delays, only a small fraction of currently existing hardware neuromorphic systems supports them. In this paper we demonstrate a strategy to implement temporal delays in hardware spiking neural networks distributed across multiple Very Large Scale Integration (VLSI) chips. This is achieved by exploiting the inherent device mismatch present in the analog circuits that implement silicon neurons and synapses inside the chips, and the digital communication infrastructure used to configure the network topology and transmit the spikes across chips. We present an example of a recurrent VLSI spiking neural network that employs axonal delays and demonstrate how the proposed strategy efficiently implements them in hardware.

Background Efficient perception of attacking pathogens is essential for plants. Plant defense is evoked by molecules termed elicitors. Endogenous elicitors or damage-associated molecular patterns (DAMPs) originate from plant materials upon injury or pathogen activity. While there are comparably well-characterized examples for DAMPs, often oligogalacturonides (OGAs), generated by the activity of fungal pathogens, endogenous elicitors evoked by bacterial pathogens have been rarely described. In particular, the signal perception and transduction processes involved in DAMP generation are poorly characterized. Results A mutant strain of the phytopathogenic bacterium Xanthomonas campestris pv. campestris deficient in exbD2, which encodes a component of its unusual elaborate TonB system, had impaired pectate lyase activity and caused no visible symptoms for defense on the non-host plant pepper (Capsicum annuum). A co-incubation of X. campestris pv. campestris with isolated cell wall material from C. annuum led to the release of compounds which induced an oxidative burst in cell suspension cultures of the non-host plant. Lipopolysaccharides and proteins were ruled out as elicitors by polymyxin B and heat treatment, respectively. After hydrolysis with trifluoroacetic acid and subsequent HPAE chromatography, the elicitor preparation contained galacturonic acid, the monosaccharide constituent of pectate. OGAs were isolated from this crude elicitor preparation by HPAEC and tested for their biological activity. While small OGAs were unable to induce an oxidative burst, the elicitor activity in cell suspension cultures of the non-host plants tobacco and pepper increased with the degree of polymerization (DP). Maximal elicitor activity was observed for DPs exceeding 8. In contrast to the X. campestris pv. campestris wild type B100, the exbD2 mutant was unable to generate elicitor activity from plant cell wall material or from pectin. Conclusions To our knowledge, this is the second report on a DAMP generated by bacterial features. The generation of the OGA elicitor is embedded in a complex exchange of signals within the framework of the plant-microbe interaction of C. annuum and X. campestris pv. campestris. The bacterial TonB-system is essential for the substrate-induced generation of extracellular pectate lyase activity. This is the first demonstration that a TonB-system is involved in bacterial trans-envelope signaling in the context of a pathogenic interaction with a plant.

Genome-wide analyses of repetitive DNA suggest a significant impact particularly of transposable elements on genome size and evolution of virtually all eukaryotic organisms. In this study, we analyzed the abundance and diversity of the hAT transposon superfamily of the sugar beet (B. vulgaris) genome, using molecular, bioinformatic and cytogenetic approaches. We identified 81 transposase-coding sequences, three of which are part of structurally intact but nonfunctional hAT transposons (BvhAT), in a B. vulgaris BAC library as well as in whole genome sequencing-derived data sets. Additionally, 116 complete and 497 truncated non-autonomous BvhAT derivatives lacking the transposase gene were in silico-detected. The 116 complete derivatives were subdivided into four BvhATpin groups each characterized by a distinct terminal inverted repeat motif. Both BvhAT and BvhATpin transposons are specific for species of the genus Beta and closely related species, showing a localization on B. vulgaris chromosomes predominantely in euchromatic regions. The lack of any BvhAT transposase function together with the high degree of degeneration observed for the BvhAT and the BvhATpin genomic fraction contrasts with the abundance and activity of autonomous and non-autonomous hAT transposons revealed in other plant species. This indicates a possible genus-specific structural and functional repression of the hAT transposon superfamily during Beta diversification and evolution.

Background The distance between two genomes is often computed by comparing only the common markers between them. Some approaches are also able to deal with non-common markers, allowing the insertion or the deletion of such markers. In these models, a deletion and a subsequent insertion that occur at the same position of the genome count for two sorting steps. Results Here we propose a new model that sorts non-common markers with substitutions, which are more powerful operations that comprehend insertions and deletions. A deletion and an insertion that occur at the same position of the genome can be modeled as a substitution, counting for a single sorting step. Conclusions Comparing genomes with unequal content, but without duplicated markers, we give a linear time algorithm to compute the genomic distance considering substitutions and double-cut-and-join (DCJ) operations. This model provides a parsimonious genomic distance to handle genomes free of duplicated markers, that is in practice a lower bound to the real genomic distances. The method could also be used to refine orthology assignments, since in some cases a substitution could actually correspond to an unannotated orthology.

Background Classical approaches to compute the genomic distance are usually limited to genomes with the same content, without duplicated markers. However, differences in the gene content are frequently observed and can reflect important evolutionary aspects. A few polynomial time algorithms that include genome rearrangements, insertions and deletions (or substitutions) were already proposed. These methods often allow a block of contiguous markers to be inserted, deleted or substituted at once but result in distance functions that do not respect the triangular inequality and hence do not constitute metrics. Results In the present study we discuss the disruption of the triangular inequality in some of the available methods and give a framework to establish an efficient correction for two models recently proposed, one that includes insertions, deletions and double cut and join (DCJ) operations, and one that includes substitutions and DCJ operations. Conclusions We show that the proposed framework establishes the triangular inequality in both distances, by summing a surcharge on indel operations and on substitutions that depends only on the number of markers affected by these operations. This correction can be applied a posteriori, without interfering with the already available formulas to compute these distances. We claim that this correction leads to distances that are biologically more plausible.