Arabidopsis Root Microbiome Microfluidic (ARMM) Device for Imaging Bacterial Colonization and Morphogenesis of Arabidopsis Roots.

Imaging the spatiotemporal dynamics of host-microbiota interactions is of particular interest for augmenting our understanding of these complex systems. This is especially true of plant-microbe interactions happening around, on, and inside plant roots where relatively little is understood about the dynamics of these systems. Over the past decade, a number of microfluidic devices have been developed to grow plants hydroponically in gnotobiotic conditions and image morphogenesis of the root and/or dynamics with fluorescently labeled bacteria from the plant root microbiome. Here we describe the construction and use of our Arabidopsis Root Microbiome Microfluidic (ARMM) device for imaging fluorescent protein expressing bacteria and their colonization of Arabidopsis roots. In contrast to other plant root imaging devices, we designed this device to have a larger chamber for observing Arabidopsis root elongation and plant-microbe interactions with older seedlings (between 1.5 and 4 weeks after germination) and a 200 µm chamber depth to specifically maintain thin Arabidopsis roots within the focal distance of the confocal microscope. Our device incorporates a new approach to growing Arabidopsis seedlings in screw-top tube caps for simplified germination and transfer to the device. We present representative images from the ARMM device including high resolution cross section images of bacterial colonization at the root surface.

Comprehensive assessment of blood-brain barrier opening and sterile inflammatory response: unraveling the therapeutic window.

Microbubbles (MBs) combined with focused ultrasound (FUS) has emerged as a promising noninvasive technique to permeabilize the blood-brain barrier (BBB) for drug delivery into the brain. However, the safety and biological consequences of BBB opening (BBBO) remain incompletely understood. This study aims to investigate the effects of two parameters mediating BBBO: microbubble volume dose (MVD) and mechanical index (MI). High-resolution MRI-guided FUS was employed in mouse brains to assess BBBO by manipulating these two parameters. Afterward, the sterile inflammatory response (SIR) was studied 6 h post-FUS treatment. Results demonstrated that both MVD and MI significantly influenced the extent of BBBO, with higher MVD and MI leading to increased permeability. Moreover, RNA sequencing revealed upregulation of major inflammatory pathways and immune cell infiltration after BBBO, indicating the presence and extent of SIR. Gene set enrichment analysis identified 12 gene sets associated with inflammatory responses that were significantly upregulated at higher MVD or MI. A therapeutic window was established between therapeutically relevant BBBO and the onset of SIR, providing operating regimes to avoid damage from stimulation of the NFκB pathway via TNFɑ signaling to apoptosis. These results contribute to the optimization and standardization of BBB opening parameters for safe and effective drug delivery to the brain and further elucidate the underlying molecular mechanisms driving sterile inflammation.

Effect of Microbubble Size, Composition and Multiple Sonication Points on Sterile Inflammatory Response in Focused Ultrasound-Mediated Blood-Brain Barrier Opening.

Blood-brain barrier opening (BBBO) using focused ultrasound (FUS) and microbubbles (MBs) has emerged as a promising technique for delivering therapeutics to the brain. However, the influence of various FUS and MB parameters on BBBO and subsequent sterile inflammatory response (SIR) remains unclear. In this study, we investigated the effects of MB size and composition, as well as the number of FUS sonication points, on BBBO and SIR in an immunocompetent mouse model. Using MRI-guided MB+FUS, we targeted the striatum and assessed extravasation of an MRI contrast agent to assess BBBO and RNAseq to assess SIR. Our results revealed distinct effects of these parameters on BBBO and SIR. Specifically, at a matched microbubble volume dose (MVD), MB size did not affect the extent of BBBO, but smaller (1 µm diameter) MBs exhibited a lower classification of SIR than larger (3 or 5 µm diameter) MBs. Lipid-shelled microbubbles exhibited greater BBBO and a more pronounced SIR compared to albumin-shelled microbubbles, likely owing to the latter's poor in vivo stability. As expected, increasing the number of sonication points resulted in greater BBBO and SIR. Furthermore, correlation analysis revealed strong associations between passive cavitation detection measurements of harmonic and inertial MB echoes, BBBO and the expression of SIR gene sets. Our findings highlight the critical role of MB and FUS parameters in modulating BBBO and subsequent SIR in the brain. These insights inform the development of targeted drug delivery strategies and the mitigation of adverse inflammatory reactions in neurological disorders.

Targeting diffuse midline gliomas: The promise of focused ultrasound-mediated blood-brain barrier opening.

Diffuse midline gliomas (DMGs), including diffuse intrinsic pontine glioma, have among the highest mortality rates of all childhood cancers, despite recent advancements in cancer therapeutics. This is partly because, unlike some CNS tumors, the blood-brain barrier (BBB) of DMG tumor vessels remains intact. The BBB prevents the permeation of many molecular therapies into the brain parenchyma, where the cancer cells reside. Focused ultrasound (FUS) with microbubbles has recently emerged as an innovative and exciting technology that non-invasively permeabilizes the BBB in a small focal region with millimeter precision. In this review, current treatment methods and biological barriers to treating DMGs are discussed. State-of-the-art FUS-mediated BBB opening is then examined, with a focus on the effects of various ultrasound parameters and the treatment of DMGs.

Messenger RNA enrichment using synthetic oligo(T) click nucleic acids.

Enrichment of mRNA is a key step in a number of molecular biology techniques, particularly in the rapidly growing field of transcriptomics. Currently, mRNA is isolated using oligo(thymine) DNA (oligo(dT)) immobilized on solid supports, which binds to the poly(A) tail of mRNA to pull the mRNA out of solution through the use of magnets or centrifugal filters. Here, a simple method to isolate mRNA by complexing it with synthetic click nucleic acids (CNAs) is described. Oligo(T) CNA bound efficiently to mRNA, and because of the insolubility of CNA in water, >90% of mRNA was readily removed from solution using this method. Simple washing, buffer exchange, and heating steps enabled mRNA's enrichment from total RNA, with a yield of 3.1 ± 1.5% of the input total RNA by mass, comparable to the yield from commercially available mRNA enrichment beads. Further, the integrity and activity of mRNA after CNA-facilitated pulldown and release was evaluated through two assays. In vitro translation of EGFP mRNA confirmed the translatability of mRNA into functional protein and RT-qPCR was used to amplify enriched mRNA from total RNA extracts and compare gene expression to results obtained using commercially available products.