Release of insulin granules by simultaneous, high-speed correlative SICM-FCM

SummaryExocytosis of peptides and steroids stored in a dense core vesicular (DCV) form is the final step of every secretory pathway, indispensable for the function of nervous, endocrine and immune systems. The lack of live imaging techniques capable of direct, label-free visualisation of DCV release makes many aspects of the exocytotic process inaccessible to investigation. We describe the application of correlative scanning ion conductance and fluorescence confocal microscopy (SICM-FCM) to study the exocytosis of individual granules of insulin from the top, non-adherent, surface of pancreatic β-cells. Using SICM-FCM, we were first to directly follow the topographical changes associated with physiologically-induced release of insulin DCVs. This allowed us to report the kinetics of the full fusion of the insulin vesicle as well as the subsequent solubilisation of the released insulin crystal.

Direct Label Free Detection of Orchid Virus Using a Micro/Nano Hybrid Structured Biosensor

In this study, a label-free detection approach for effective detection of the odontoglossum ringspot virus (ORSV) infected orchids has been developed. We used semiconductor fabrication process to fabricate 1,810 micro/nano hybrid structured sensing electrodes on a 8 inch reclaimed wafer. The self-assembled monolayer (SAM) process was then employed to sequentially modify the electrode surface with 11-mercaptoundecanoic acid (11-MUA), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)/ N-hydroxysuccinimide (NHS), anti-ORSV, and ORSV. EIS was conducted for the ORSV concentration detection. Experimental results demonstrated that the ORSV concentration in a virus infected orchid leaf could be effectively detected. When compared with the ELISA kit, our device possesses a wider linear detection range (0.5–50,000 ng/mL) and a higher sensitivity. The specificity of our device on ORSV detection was also confirmed. Our sensing device retains advantages, such as label-free, lower amounts of the antibody and target sample required, low detection time, and a wider linear detection range. Those results imply the feasibility of our sensing device in field applications.

SMRT long reads and Direct Label and Stain optical maps allow the generation of a high-quality genome assembly for the European barn swallow (Hirundo rustica rustica)

Background The barn swallow (Hirundo rustica) is a migratory bird that has been the focus of a large number of ecological, behavioral, and genetic studies. To facilitate further population genetics and genomic studies, we present a reference genome assembly for the European subspecies (H. r. rustica). Findings As part of the Genome10K effort on generating high-quality vertebrate genomes (Vertebrate Genomes Project), we have assembled a highly contiguous genome assembly using single molecule real-time (SMRT) DNA sequencing and several Bionano optical map technologies. We compared and integrated optical maps derived from both the Nick, Label, Repair, and Stain technology and from the Direct Label and Stain (DLS) technology. As proposed by Bionano, DLS more than doubled the scaffold N50 with respect to the nickase. The dual enzyme hybrid scaffold led to a further marginal increase in scaffold N50 and an overall increase of confidence in the scaffolds. After removal of haplotigs, the final assembly is approximately 1.21 Gbp in size, with a scaffold N50 value of more than 25.95 Mbp. Conclusions This high-quality genome assembly represents a valuable resource for future studies of population genetics and genomics in the barn swallow and for studies concerning the evolution of avian genomes. It also represents one of the very first genomes assembled by combining SMRT long-read sequencing with the new Bionano DLS technology for scaffolding. The quality of this assembly demonstrates the potential of this methodology to substantially increase the contiguity of genome assemblies.

An Aptamer-Based Biosensor for Direct, Label-Free Detection of Melamine in Raw Milk

Melamine, a nitrogen-rich compound, has been used as a food and milk additive to falsely increase the protein content. However, melamine is toxic, and high melamine levels in food or in milk can cause kidney and urinary problems, or even death. Hence, the detection of melamine in food and milk is desirable, for which numerous detection methods have been developed. Several methods have successfully detected melamine in raw milk; however, they require a sample preparation before the analyses. This study aimed to develop an aptamer-DNAzyme conjugated biosensor for label-free detection of melamine, in raw milk, without any sample preparation. An aptamer-DNAzyme conjugated biosensor was developed via screening using microarray analysis to identify the candidate aptamers followed by an optimization, to reduce the background noise and improve the aptamer properties, thereby, enhancing the signal-to-noise (S/N) ratio of the screened biosensor. The developed biosensor was evaluated via colorimetric detection and tested with raw milk without any sample preparation, using N-methylmesoporphyrin IX for fluorescence detection. The biosensor displayed significantly higher signal intensity at 2 mM melamine (S/N ratio, 20.2), which was sufficient to detect melamine at high concentrations, in raw milk.

SMRT long reads and Direct Label and Stain optical maps allow the generation of a high-quality genome assembly for the European barn swallow (Hirundo rustica rustica)

ABSTRACTBackgroundThe barn swallow (Hirundo rustica) is a migratory bird that has been the focus of a large number of ecological, behavioural and genetic studies. To facilitate further population genetics and genomic studies, here we present a reference genome assembly for the European subspecies (H. r. rustica).FindingsAs part of the Genome10K (G10K) effort on generating high quality vertebrate genomes, we have assembled a highly contiguous genome assembly using Single Molecule Real-Time (SMRT) DNA sequencing and several Bionano optical map technologies. We compared and integrated optical maps derived both from the Nick, Label, Repair and Stain and from the Direct Label and Stain (DLS) technologies. As proposed by Bionano, the DLS more than doubled the scaffold N50 with respect to the nickase. The dual enzyme hybrid scaffold led to a further marginal increase in scaffold N50 and an overall increase of confidence in the scaffolds. After removal of haplotigs, the final assembly is approximately 1.21 Gbp in size, with a scaffold N50 value of over 25.95 Mbp.ConclusionsThis high-quality genome assembly represents a valuable resource for further studies of population genetics and genomics in the barn swallow, and for studies concerning the evolution of avian genomes. It also represents one of the very first genomes assembled by combining SMRT long-read sequencing with the new Bionano DLS technology for scaffolding. The quality of this assembly demonstrates the potential of this methodology to substantially increase the contiguity of genome assemblies.