Search

The requirement of a large input amount (500 ng) for Nanopore direct RNA‐seq presents a major challenge for low input transcriptomic analysis and early pathogen surveillance. The high RNA input requirement is attributed to significant sample loss associated with library preparation using solid‐phase reversible immobilization (SPRI) beads. A novel solid‐phase catalysis strategy for RNA library preparation to circumvent the need for SPRI bead purification to remove enzymes is reported here. This new approach leverages concurrent processing of non‐polyadenylated transcripts with immobilized poly(A) polymerase and T4 DNA ligase, followed by directly loading the prepared library onto a flow cell. Whole transcriptome sequencing, using a human pathogen Listeria monocytogenes as a model, demonstrates this new method displays little sample loss, takes much less time, and generates higher sequencing throughput correlated with reduced nanopore fouling compared to the current library preparation for 500 ng input. Consequently, this approach enables Nanopore low‐input direct RNA‐seq, improving pathogen detection and transcript identification in a microbial community standard with spike‐in transcript controls. Besides, as evident in the bioinformatic analysis, the new method provides accurate RNA consensus with high fidelity and identifies higher numbers of expressed genes for both high and low input RNA amounts. [ABSTRACT FROM AUTHOR]

Summary: Quorum sensing (QS) exists widely among bacteria, enabling a transition to multicellular behaviour after bacterial populations reach a particular density. The coordination of multicellularity enables biotechnological application, dissolution of biofilms, coordination of virulence, and so forth. Here, a method to elicit and subsequently disperse multicellular behaviour among QS‐negative cells is developed using magnetic nanoparticle assembly. We fabricated magnetic nanoparticles (MNPs, ∼5 nm) that electrostatically collect wild‐type (WT) Escherichia coli BL21 cells and brings them into proximity of bioengineered E. coli [CT104 (W3110 lsrFG− luxS− pCT6 + pET‐DsRed)] reporter cells that exhibit a QS response after receiving autoinducer‐2 (AI‐2). By shortening the distance between WT and reporter cells (e.g., increasing local available AI‐2 concentrations), the QS response signalling was amplified four‐fold compared to that in native conditions without assembly. This study suggests potential applications in facilitating intercellular communication and modulating multicellular behaviours based on user‐specified designs. [ABSTRACT FROM AUTHOR]

Sensor-based chemical analyses commonly enlist either the molecular recognition capabilities of biology (e.g., enzyme biosensors) or advanced information processing algorithms (e.g., the electronic nose). Here, a hybrid approach is proposed in which an enzyme is used to 'filter' chemical information and write this information to a film which then serves as a permanent storage medium that can be 'read' repeatedly, interactively, and by multiple sensor modalities. This approach is demonstrated by analyzing common dietary phenols that are reported to offer health benefits. Specifically, the enzyme tyrosinase is used to convert these phenols into reactive quinones that graft (i.e., write) to a chitosan film. Grafting can be detected by optical, mechanical, and electrochemical sensors. Importantly, grafting confers redox activity to the films and this redox activity can be probed interactively by advanced electrochemical methods that allow the intrinsic redox reactivities to be compared, redox interactions to be identified, and biologically relevant redox activities to be examined. The transfer of chemical and biological information to a film is envisioned to provide broader access to the extensive capabilities offered by sensor technologies and signal processing methodologies. [ABSTRACT FROM AUTHOR]