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Wednesday, October 23, 2019

Molecular barcoding of viral vectors enables mapping and optimization of mRNA trans-splicing.

Genome editing has proven to be highly potent in the generation of functional gene knockouts in dividing cells. In the CNS however, efficient technologies to repair sequences are yet to materialize. Reprogramming on the mRNA level is an attractive alternative as it provides means to perform in situ editing of coding sequences without nuclease dependency. Furthermore, de novo sequences can be inserted without the requirement of homologous recombination. Such reprogramming would enable efficient editing in quiescent cells (e.g., neurons) with an attractive safety profile for translational therapies. In this study, we applied a novel molecular-barcoded screening assay to investigate RNA…

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Wednesday, October 23, 2019

An online bioinformatics tool predicts zinc finger and TALE nuclease off-target cleavage.

Although engineered nucleases can efficiently cleave intracellular DNA at desired target sites, major concerns remain on potential ‘off-target’ cleavage that may occur throughout the genome. We developed an online tool: predicted report of genome-wide nuclease off-target sites (PROGNOS) that effectively identifies off-target sites. The initial bioinformatics algorithms in PROGNOS were validated by predicting 44 of 65 previously confirmed off-target sites, and by uncovering a new off-target site for the extensively studied zinc finger nucleases (ZFNs) targeting C-C chemokine receptor type 5. Using PROGNOS, we rapidly interrogated 128 potential off-target sites for newly designed transcription activator-like effector nucleases containing either Asn-Asn…

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Sunday, September 22, 2019

Discovery of a divergent HPIV4 from respiratory secretions using second and third generation metagenomic sequencing.

Molecular detection of viruses has been aided by high-throughput sequencing, permitting the genomic characterization of emerging strains. In this study, we comprehensively screened 500 respiratory secretions from children with upper and/or lower respiratory tract infections for viral pathogens. The viruses detected are described, including a divergent human parainfluenza virus type 4 from GS FLX pyrosequencing of 92 specimens. Complete full-genome characterization of the virus followed, using Single Molecule, Real-Time (SMRT) sequencing. Subsequent “primer walking” combined with Sanger sequencing validated the RS platform’s utility in viral sequencing from complex clinical samples. Comparative genomics reveals the divergent strain clusters with the only…

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Sunday, September 22, 2019

A comprehensive fungi-specific 18S rRNA gene sequence primer toolkit suited for diverse research issues and sequencing platforms.

Several fungi-specific primers target the 18S rRNA gene sequence, one of the prominent markers for fungal classification. The design of most primers goes back to the last decades. Since then, the number of sequences in public databases increased leading to the discovery of new fungal groups and changes in fungal taxonomy. However, no reevaluation of primers was carried out and relevant information on most primers is missing. With this study, we aimed to develop an 18S rRNA gene sequence primer toolkit allowing an easy selection of the best primer pair appropriate for different sequencing platforms, research aims (biodiversity assessment versus…

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Sunday, September 22, 2019

Analysis of aquaporins from the euryhaline barnacle Balanus improvisus reveals differential expression in response to changes in salinity.

Barnacles are sessile macro-invertebrates, found along rocky shores in coastal areas worldwide. The euryhaline bay barnacle Balanus improvisus (Darwin, 1854) (= Amphibalanus improvisus) can tolerate a wide range of salinities, but the molecular mechanisms underlying the osmoregulatory capacity of this truly brackish species are not well understood. Aquaporins are pore-forming integral membrane proteins that facilitate transport of water, small solutes and ions through cellular membranes, and that have been shown to be important for osmoregulation in many organisms. The knowledge of the function of aquaporins in crustaceans is, however, limited and nothing is known about them in barnacles. We here…

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Sunday, September 22, 2019

Alternative splice variants of AID are not stoichiometrically present at the protein level in chronic lymphocytic leukemia

Activation-induced deaminase (AID) is a DNA-mutating enzyme that mediates class-switch recombination as well as somatic hypermutation of antibody genes in B cells. Due to off-target activity, AID is implicated in lymphoma development by introducing genome-wide DNA damage and initiating chromosomal translocations such as c-myc/IgH. Several alternative splice transcripts of AID have been reported in activated B cells as well as malignant B cells such as chronic lymphocytic leukemia (CLL). As most commercially available antibodies fail to recognize alternative splice variants, their abundance in vivo, and hence their biological significance, has not been determined. In this study, we assessed the protein…

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Sunday, September 22, 2019

Multiscale patterns and drivers of arbuscular mycorrhizal fungal communities in the roots and root-associated soil of a wild perennial herb.

Arbuscular mycorrhizal (AM) fungi form diverse communities and are known to influence above-ground community dynamics and biodiversity. However, the multiscale patterns and drivers of AM fungal composition and diversity are still poorly understood. We sequenced DNA markers from roots and root-associated soil from Plantago lanceolata plants collected across multiple spatial scales to allow comparison of AM fungal communities among neighbouring plants, plant subpopulations, nearby plant populations, and regions. We also measured soil nutrients, temperature, humidity, and community composition of neighbouring plants and nonAM root-associated fungi. AM fungal communities were already highly dissimilar among neighbouring plants (c. 30 cm apart), albeit with a…

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Sunday, September 22, 2019

Single-cell mRNA isoform diversity in the mouse brain.

Alternative mRNA isoform usage is an important source of protein diversity in mammalian cells. This phenomenon has been extensively studied in bulk tissues, however, it remains unclear how this diversity is reflected in single cells.Here we use long-read sequencing technology combined with unique molecular identifiers (UMIs) to reveal patterns of alternative full-length isoform expression in single cells from the mouse brain. We found a surprising amount of isoform diversity, even after applying a conservative definition of what constitutes an isoform. Genes tend to have one or a few isoforms highly expressed and a larger number of isoforms expressed at a…

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Sunday, September 22, 2019

Discovery of enzymes for toluene synthesis from anoxic microbial communities.

Microbial toluene biosynthesis was reported in anoxic lake sediments more than three decades ago, but the enzyme catalyzing this biochemically challenging reaction has never been identified. Here we report the toluene-producing enzyme PhdB, a glycyl radical enzyme of bacterial origin that catalyzes phenylacetate decarboxylation, and its cognate activating enzyme PhdA, a radical S-adenosylmethionine enzyme, discovered in two distinct anoxic microbial communities that produce toluene. The unconventional process of enzyme discovery from a complex microbial community (>300,000 genes), rather than from a microbial isolate, involved metagenomics- and metaproteomics-enabled biochemistry, as well as in vitro confirmation of activity with recombinant enzymes. This…

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Sunday, September 22, 2019

The microbiota of freshwater fish and freshwater niches contain omega-3 producing Shewanella species.

Approximately 30 years ago, it was discovered that free-living bacteria isolated from cold ocean depths could produce polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA) (20:5n-3) or docosahexaenoic acid (DHA) (22:6n-3), two PUFA essential for human health. Numerous laboratories have also discovered that EPA- and/or DHA-producing bacteria, many of them members of the Shewanella genus, could be isolated from the intestinal tracts of omega-3 fatty acid-rich marine fish. If bacteria contribute omega-3 fatty acids to the host fish in general or if they assist some bacterial species in adaptation to cold, then cold freshwater fish or habitats should also…

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Sunday, September 22, 2019

Cell-type-specific splicing of Piezo2 regulates mechanotransduction.

Piezo2 is a mechanically activated ion channel required for touch discrimination, vibration detection, and proprioception. Here, we discovered that Piezo2 is extensively spliced, producing different Piezo2 isoforms with distinct properties. Sensory neurons from both mice and humans express a large repertoire of Piezo2 variants, whereas non-neuronal tissues express predominantly a single isoform. Notably, even within sensory ganglia, we demonstrate the splicing of Piezo2 to be cell type specific. Biophysical characterization revealed substantial differences in ion permeability, sensitivity to calcium modulation, and inactivation kinetics among Piezo2 splice variants. Together, our results describe, at the molecular level, a potential mechanism by which…

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Sunday, September 22, 2019

Assessment of an organ-specific de novo transcriptome of the nematode trap-crop, Solanum sisymbriifolium

Solanum sisymbriifolium, also known as “Litchi Tomato” or “Sticky Nightshade,” is an undomesticated and poorly researched plant related to potato and tomato. Unlike the latter species, S. sisymbriifolium induces eggs of the cyst nematode, Globodera pallida, to hatch and migrate into its roots, but then arrests further nematode maturation. In order to provide researchers with a partial blueprint of its genetic make-up so that the mechanism of this response might be identified, we used single molecule real time (SMRT) sequencing to compile a high quality de novo transcriptome of 41,189 unigenes drawn from individually sequenced bud, root, stem, and leaf…

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Sunday, September 22, 2019

The genome sequence of the commercially cultivated mushroom Agrocybe aegerita reveals a conserved repertoire of fruiting-related genes and a versatile suite of biopolymer-degrading enzymes.

Agrocybe aegerita is an agaricomycete fungus with typical mushroom features, which is commercially cultivated for its culinary use. In nature, it is a saprotrophic or facultative pathogenic fungus causing a white-rot of hardwood in forests of warm and mild climate. The ease of cultivation and fructification on solidified media as well as its archetypal mushroom fruit body morphology render A. aegerita a well-suited model for investigating mushroom developmental biology.Here, the genome of the species is reported and analysed with respect to carbohydrate active genes and genes known to play a role during fruit body formation. In terms of fruit body…

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Sunday, September 22, 2019

Functional metagenomics reveals a novel carbapenem-hydrolyzing mobile beta-lactamase from Indian river sediments contaminated with antibiotic production waste.

Evolution has provided environmental bacteria with a plethora of genes that give resistance to antibiotic compounds. Under anthropogenic selection pressures, some of these genes are believed to be recruited over time into pathogens by horizontal gene transfer. River sediment polluted with fluoroquinolones and other drugs discharged from bulk drug production in India constitute an environment with unprecedented, long-term antibiotic selection pressures. It is therefore plausible that previously unknown resistance genes have evolved and/or are promoted here. In order to search for novel resistance genes, we therefore analyzed such river sediments by a functional metagenomics approach. DNA fragments providing resistance to…

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Sunday, September 22, 2019

Plasmid-encoded transferable mecB-mediated methicillin resistance in Staphylococcus aureus.

During cefoxitin-based nasal screening, phenotypically categorized methicillin-resistant Staphylococcus aureus (MRSA) was isolated and tested negative for the presence of the mecA and mecC genes as well as for the SCCmec-orfX junction region. The isolate was found to carry a mecB gene previously described for Macrococcus caseolyticus but not for staphylococcal species. The gene is flanked by ß-lactam regulatory genes similar to mecR, mecI, and blaZ and is part of an 84.6-kb multidrug-resistance plasmid that harbors genes encoding additional resistances to aminoglycosides (aacA-aphD, aphA, and aadK) as well as macrolides (ermB) and tetracyclines (tetS). This further plasmidborne ß-lactam resistance mechanism harbors…

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