Arrayed in vivo barcoding for multiplexed sequence verification of plasmid DNA and demultiplexing of pooled libraries.

Sequence verification of plasmid DNA is critical for many cloning and molecular biology workflows. To leverage high-throughput sequencing, several methods have been developed that add a unique DNA barcode to individual samples prior to pooling and sequencing. However, these methods require an individual plasmid extraction and/or in vitro barcoding reaction for each sample processed, limiting throughput and adding cost.

Arrayed barcoding for multiplexed sequence verification of plasmid DNA and demultiplexing of pooled libraries.

Sequence verification of plasmid DNA is critical for many cloning and molecular biology workflows. To leverage high-throughput sequencing, several methods have been developed that add a unique DNA barcode to individual samples prior to pooling and sequencing. However, these methods require an individual plasmid extraction and/or barcoding reaction for each sample processed, limiting throughput and adding cost.

The unrestricted global effort to complete the COOL trial.

Background: Severe complicated intra-abdominal sepsis (SCIAS) has an increasing incidence with mortality rates over 80% in some settings. Mortality typically results from disruption of the gastrointestinal tract, progressive and self-perpetuating bio-mediator generation, systemic inflammation, and multiple organ failure. A further therapeutic option may be open abdomen (OA) management with negative peritoneal pressure therapy (NPPT) to remove inflammatory ascites and attenuate the systemic damage from SCIAS, although there are definite risks of leaving the abdomen open whenever it might possibly be closed.

Fungicide sensitivity of grapevine bacteria with plant growth-promoting traits and antagonistic activity as non-target microorganisms.

This study evaluates the capacity of commercial formulations of synthetic fungicides to inhibit grapevine bacterial growth when sprayed on vineyards to control diseases, such as downy mildew, powdery mildew and secondary rots. Fungicide sensitivity plate assays were carried out on bacteria isolated from vineyards that were also identified and characterized for their plant growth-promoting (PGP) traits and antifungal activity. The high taxonomic variability of bacteria screened with different chemical classes of fungicides is one new finding of this study.

Assembly of Long-Adapter Single-Strand Oligonucleotide (LASSO) Probes for Massively Parallel Capture of Kilobase Size DNA Targets.

Genome DNA sequencing has become an affordable means to resolve questions about the genetic background of life. However, the biological functions of many DNA-encoded sequences are still relatively unknown. A highly scalable and cost-effective cloning method to select natural DNA targets from genomic templates is therefore urgently needed to enable rapid understanding of the biological products of genomes.

Massively parallel DNA target capture using long adapter single stranded oligonucleotide (LASSO) probes assembled through a novel DNA recombinase mediated methodology.

In the attempt to bridge the widening gap from DNA sequence to biological function, we developed a novel methodology to assemble Long-Adapter Single-Strand Oligonucleotide (LASSO) probe libraries that enabled the massively multiplexed capture of kilobase-sized DNA fragments for downstream long read DNA sequencing or expression. This method uses short DNA oligonucleotides (pre-LASSO probes) and a plasmid vector that supplies the linker sequence for the mature LASSO probe through Cre-LoxP intramolecular recombination. This strategy generates high quality LASSO probes libraries (≈46% of correct probes).

Quantitative assessment of LASSO probe assembly and long-read multiplexed cloning.

Background: Long Adapter Single-Stranded Oligonucleotide (LASSO) probes were developed as a novel tool for massively parallel cloning of kilobase-long genomic DNA sequences. LASSO dramatically improves the capture length limit of current DNA padlock probe technology from approximately 150 bps to several kbps. High-throughput LASSO capture involves the parallel assembly of thousands of probes.

Computational Simulation of Adapter Length-Dependent LASSO Probe Capture Efficiency.

Multiplexed cloning of long DNA sequences is a valuable technique in many biotechnology applications, such as long-read genome sequencing and the creation of open reading frame (ORF) libraries. Long-adapter single-stranded oligonucleotide (LASSO) probes have shown promise as a tool to clone long DNA fragments. LASSO probes are molecular inversion probes (MIP) engineered with an adapter region of user-defined length, flanked between template-specific probe sequences.

Long-adapter single-strand oligonucleotide probes for the massively multiplexed cloning of kilobase genome regions.

As the catalogue of sequenced genomes and metagenomes continues to grow, massively parallel approaches for the comprehensive and functional analysis of gene products and regulatory elements are becoming increasingly valuable. Current strategies to synthesize or clone complex libraries of DNA sequences are limited by the length of the DNA targets, throughput and cost. Here, we show that long-adapter single-strand oligonucleotide (LASSO) probes can capture and clone thousands of kilobase DNA fragments in a single reaction.

Transcriptional Reprogramming of the Mycoparasitic Fungus Ampelomyces quisqualis During the Powdery Mildew Host-Induced Germination.

Ampelomyces quisqualis is a mycoparasite of a diverse range of phytopathogenic fungi associated with the powdery mildew disease. Among them are several Erysiphaceae species with great economic impact on high-value crops such as grape. Due to its ability to parasitize and prevent the spread of powdery mildews, A.

Intra- and interlaboratory performances of two commercial antimicrobial susceptibility testing methods for bifidobacteria and nonenterococcal lactic acid bacteria.

In a small-scale harmonization study involving nine laboratories in eight European countries, the intra- and interlaboratory performances of two commercially available systems, i.e., the VetMIC microplate system and Etest, for antimicrobial susceptibility testing of nonenterococcal lactic acid bacteria (NELAB) and bifidobacteria were analyzed.

Susceptibility of Lactobacillus plantarum strains to six antibiotics and definition of new susceptibility-resistance cutoff values.

The minimum inhibitory concentrations (MICs) of six antibiotics with activity against gram-positive bacteria (ampicillin, clindamycin, erythromycin, gentamicin, streptomycin, and tetracycline) were determined by microdilution and the Etest in 121 Lactobacillus plantarum strains of plant and dairy origin. MIC values for all antibiotics varied widely between strains. The analysis of both absolute MICs and their distribution was used to define new susceptibility-resistance cutoff values for all antibiotics, except for streptomycin.

Susceptibility of Streptococcus thermophilus to antibiotics.

A total of 74 Streptococcus thermophilus isolates collected between 1948 and 2005 from different environments were investigated to assess erythromycin, clindamycin, streptomycin, gentamicin, tetracycline and ampicillin susceptibility by means of microdilution, Etest and disk diffusion methods. For this purpose a new S. thermophilus Susceptibility test Medium (SSM) was developed.