Highly efficient and inducible DNA excision in transgenic silkworms using the FLP/<Emphasis Type="Italic">FRT</Emphasis> site-specific recombination system

Efficient and inducible recombinase-mediated DNA excision is an optimal technology for automatically deleting unwanted DNA sequences, including selection marker genes. However, this methodology has yet to be established in transgenic silkworms. To achieve efficient and inducible FLP recombinase-mediated DNA excision in transgenic silkworms, one transgenic target strain (TTS) containing an FRT-flanked silkworm cytoplasmic actin 3 gene promoter (A3)-enhanced green fluorescent protein (EGFP) expression cassette, as well as two different types of FLP recombinase expression helper strains were generated. Then, the FLP recombinase was introduced into the TTS silkworms by pre-blastoderm microinjection and sexual hybridization. Successful recombinase-mediated deletion of the A3-EGFP expression cassette was observed in the offspring of the TTS, and the excision efficiencies of the FLP expression vector and FLP mRNA pre-blastoderm microinjection were 2.38 and 13.3 %, respectively. The excision efficiencies resulting from hybridization between the TTS and the helper strain that contained a heat shock protein 70 (Hsp70)-FLP expression cassette ranged from 32.14 to 36.67 % after heat shock treatment, while the excision efficiencies resulting from hybridization between the TTS and the helper strain containing the A3-FLP expression cassette ranged from 97.01 to 100 %. These results demonstrate that the FLP/FRT system can be used to achieve highly efficient and inducible post-integration excision of unwanted DNA sequences in transgenic silkworms in vivo. Our present study will facilitate the development and application of the FLP/FRT system for the functional analysis of unknown genes, and establish the safety of transgenic technologies in the silkworm and other lepidopteran species.     

Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area

PacBio RS II is the first commercialized third-generation DNA sequencer able to sequence a single molecule DNA in real-time without amplification. PacBio RS II’s sequencing technology is novel and unique, enabling the direct observation of DNA synthesis by DNA polymerase. PacBio RS II confers four major advantages compared to other sequencing technologies: long read lengths, high consensus accuracy, a low degree of bias, and simultaneous capability of epigenetic characterization. These advantages surmount the obstacle of sequencing genomic regions such as high/low G+C, tandem repeat, and interspersed repeat regions. Moreover, PacBio RS II is ideal for whole genome sequencing, targeted sequencing, complex population analysis, RNA sequencing, and epigenetics characterization. With PacBio RS II, we have sequenced and analyzed the genomes of many species, from viruses to humans. Herein, we summarize and review some of our key genome sequencing projects, including full-length viral sequencing, complete bacterial genome and almost-complete plant genome assemblies, and long amplicon sequencing of a disease-associated gene region. We believe that PacBio RS II is not only an effective tool for use in the basic biological sciences but also in the medical/clinical setting.     

CRISPR/Cas9-mediated efficient targeted mutagenesis has the potential to accelerate the domestication of <Emphasis Type="Italic">Coffea canephora</Emphasis>

Genome editing, which is an unprecedented technological breakthrough, has provided a valuable means of creating targeted mutations in plant genomes. In this study, we developed a genomic web tool to identify all gRNA target sequences in the coffee genome, along with potential off-targets. In all, 8,145,748 CRISPR guides were identified in the draft genome of Coffea canephora corresponding to 5,338,568 different sequences and, of these, 4,655,458 were single, and 514,591 were covering exons. The proof of concept was established by targeting the phytoene desaturase gene (CcPDS) using the Agrobacterium tumefaciens transformation technique and somatic embryogenesis as the plant regeneration method. An analysis of the RNA-guided genome-editing events showed that 22.8% of the regenerated plants were heterozygous mutants and 7.6% were homozygous mutants. Mutation efficiency at the target site was estimated to be 30.4%. We demonstrated that genome editing by the CRISPR/Cas9 method is an efficient and reliable way of knocking out genes of agronomic interest in the coffee tree, opening up the way for coffee molecular breeding. Our results also showed that the use of somatic embryogenesis, as the method for regenerating genome-edited plants, could restrict the choice of targeted genes to those that are not essential to the embryo development and germination steps.     

Binding and segmentation of multiple objects through neural oscillators inhibited by contour information

 Temporal correlation of neuronal activity has been suggested as a criterion for multiple object recognition. In this work, a two-dimensional network of simplified Wilson-Cowan oscillators is used to manage the binding and segmentation problem of a visual scene according to the connectedness Gestalt criterion. Binding is achieved via original coupling terms that link excitatory units to both excitatory and inhibitory units of adjacent neurons. These local coupling terms are time independent, i.e., they do not require Hebbian learning during the simulations. Segmentation is realized by a two-layer processing of the visual image. The first layer extracts all object contours from the image by means of “retinal cells” with an “on-center” receptive field. Information on contour is used to selectively inhibit Wilson-Cowan oscillators in the second layer, thus realizing a strong separation among neurons in different objects. Accidental synchronism between oscillations in different objects is prevented with the use of a global inhibitor, i.e., a global neuron that computes the overall activity in the Wilson-Cowan network and sends back an inhibitory signal. Simulations performed in a 50×50 neural grid with 21 different visual scenes (containing up to eight objects + background) with random initial conditions demonstrate that the network can correctly segment objects in almost 100% of cases using a single set of parameters, i.e., without the need to adjust parameters from one visual scene to the next. The network is robust with reference to dynamical noise superimposed on oscillatory neurons. Moreover, the network can segment both black objects on white background and vice versa and is able to deal with the problem of “fragmentation.” The main limitation of the network is its sensitivity to static noise superimposed on the objects. Overcoming this problem requires implementation of more robust mechanisms for contour enhancement in the first layer in agreement with mechanisms actually realized in the visual cortex. Received: 25 October 2001 / Accepted: 26 February 2003 / Published online: 20 May 2003 Correspondence to: Mauro Ursino (e-mail: mursino@deis.unibo.it, Tel.: +39-051-2093008, Fax: +39-051-2093073)     

B cell abnormalities in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a chronic, multisystem autoimmune disease characterized by the differentiation of short- and long-lived immunoglobulin secreting plasma cells that secrete pathogenic autoantibodies. Ectopic germinal centers and plasma cells secreting autoantibodies have been observed in lupus nephritis kidneys. Candidate genetic susceptibility loci for SLE include genes that affect differentiation and survival of plasma cells, such as those that influence activation, proliferation, cytokine and chemokine secretion/responsiveness, and apoptosis of the T and B cells that are involved in humoral immunity generated in germinal centers, as well as genes that are involved in presentation and clearance of apoptotic material and autoantigens by antigen presenting cells and other phagocytes. Emerging data have demonstrated that B lymphocytes are active participants in humoral immune responses that lead to T-dependent and T-independent differentiation of immunoglobulin-secreting plasma cells by homotypic CD154-CD40 interactions as well as continued stimulation by B cell activating factor through B cell maturation antigen, B cell activating factor receptor and transmembrane activater.     

Comparison of damage to native and exotic tallgrass prairie plants by natural enemies

We surveyed the prevalence and amount of leaf damage related to herbivory and pathogens on 12 pairs of exotic (invasive and noninvasive) and ecologically similar native plant species in tallgrass prairie to examine whether patterns of damage match predictions from the enemy release hypothesis. We also assessed whether natural enemy impacts differed in response to key environmental factors in tallgrass prairie by surveying the prevalence of rust on the dominant C₄ grass, Andropogon gerardii, and its congeneric invasive exotic C₄ grass, A. bladhii, in response to fire and nitrogen fertilization treatments. Overall, we found that the native species sustain 56.4% more overall leaf damage and 83.6% more herbivore-related leaf damage when compared to the exotic species. Moreover, we found that the invasive exotic species sustained less damage from enemies relative to their corresponding native species than the noninvasive exotic species. Finally, we found that burning and nitrogen fertilization both significantly increased the prevalence of rust fungi in the native grass, while rust fungi rarely occurred on the exotic grass. These results indicate that reduced damage from enemies may in part explain the successful naturalization of exotic species and the spread of invasive exotic species in tallgrass prairie.     

Antibody-protein interactions: benchmark datasets and prediction tools evaluation

Background  

The ability to predict antibody binding sites (aka antigenic determinants or B-cell epitopes) for a given protein is a precursor to new vaccine design and diagnostics. Among the various methods of B-cell epitope identification X-ray crystallography is one of the most reliable methods. Using these experimental data computational methods exist for B-cell epitope prediction. As the number of structures of antibody-protein complexes grows, further interest in prediction methods using 3D structure is anticipated. This work aims to establish a benchmark for 3D structure-based epitope prediction methods.     

Biomass-derived volatile fatty acid platform for fuels and chemicals

The typical biorefinery platforms are sugar, thermochemical (syngas), carbon-rich chains, and biogas platforms, each offering unique advantages and disadvantages. The sugar platform uses hexose and pentose sugars extracted or converted from plant body mass. The thermochemical (syngas) platform entails a chemical or biological conversion process using pyrolysis or gasification of plants to produce biofuels. The carbon-rich chains platform is used to produce biodiesel from long-chain fatty acids or glycerides. In the present work, we suggest a new platform using volatile fatty acids (VFAs) for the production of biofuels and biochemicals production. The VFAs are short-chain fatty acids composed mainly of acetate and butyrate in the anaerobic digestion (AD) process, which does not need sterilization, additional hydrolysis enzymes (cellulose or xylanase), or a high cost pretreatment step. VFAs are easily produced from almost all kinds of biomass with low lignin content (terrestrial, aquatic, and marine biomass) by the AD process. Considering that raw material alone constitutes 40∼80% of biofuel production costs, biofuels made from VFAs derived from waste organic biomass potentially offer significant economical advantage.