Investigation of type-I interferon dysregulation by arenaviruses : a multidisciplinary approach.

This report provides a detailed overview of the work performed for project number 130781, 'A Systems Biology Approach to Understanding Viral Hemorrhagic Fever Pathogenesis.' We report progress in five key areas: single cell isolation devices and control systems, fluorescent cytokine and transcription factor reporters, on-chip viral infection assays, molecular virology analysis of Arenavirus nucleoprotein structure-function, and development of computational tools to predict virus-host protein interactions. Although a great deal of work remains from that begun here, we have developed several novel single cell analysis tools and knowledge of Arenavirus biology that will facilitate and inform future publications and funding proposals

SEAM readouts after LPS stimulation.

<p>a) To explore SEAM readouts that mimic vascular inflammation, human microvasular endothelial cells were seeded and transferred to the coupled culture and micro-perfusion modules. b) After apical simulation with LPS for 4 hours, cells were and stained in-channel and the membrane was transferred to a glass slide to explore ICAM1 surface expression after exposure to stimulation. Scale bar = 40 μm. c) To demonstrate rapid and robust RNA isolation, inserts were removed from the architecture and transferred to off-chip RNA isolation. Statistically significant (p<0.001) differences in mRNA expression were observed in ICAM1, CXCL1, CXCL2, and CCL2 with VFW (endothelial marker) remaining constant. SEAM is able to replicate both gene expression and differential surface protein responses to LPS stimulation with an easy to use workflow.</p

Differential height vs. predicted flow rate and shear stress.

<p>Differential height vs. predicted flow rate and shear stress.</p

A Reversibly Sealed, Easy Access, Modular (SEAM) Microfluidic Architecture to Establish <i>In Vitro</i> Tissue Interfaces - Fig 3

<p>a) Layers comprising the gravity fed perfusion module. b) Side view of connected perfusion and culture modules. The fluidic circuit (inlet reservoir, aa, through the culture module to the outlet reservoir, bb) was gently primed with medium using a syringe. c) Once filled, surface tension kept the medium from spilling out of the reservoirs, and the difference in hydrostatic pressure <b>Δ</b>P induced fluid flow from inlet to outlet reservoirs.</p

A Reversibly Sealed, Easy Access, Modular (SEAM) Microfluidic Architecture to Establish <i>In Vitro</i> Tissue Interfaces

<div><p>Microfluidic barrier tissue models have emerged as advanced <i>in vitro</i> tools to explore interactions with external stimuli such as drug candidates, pathogens, or toxins. However, the procedures required to establish and maintain these systems can be challenging to implement for end users, particularly those without significant in-house engineering expertise. Here we present a module-based approach that provides an easy-to-use workflow to establish, maintain, and analyze microscale tissue constructs. Our approach begins with a removable culture insert that is magnetically coupled, decoupled, and transferred between standalone, prefabricated microfluidic modules for simplified cell seeding, culture, and downstream analysis. The modular approach allows several options for perfusion including standard syringe pumps or integration with a self-contained gravity-fed module for simple cell maintenance. As proof of concept, we establish a culture of primary human microvascular endothelial cells (HMVEC) and report combined surface protein imaging and gene expression after controlled apical stimulation with the bacterial endotoxin lipopolysaccharide (LPS). We also demonstrate the feasibility of incorporating hydrated biomaterial interfaces into the microfluidic architecture by integrating an ultra-thin (< 1 <b>μ</b>m), self-assembled hyaluronic acid/peptide amphiphile culture membrane with brain-specific Young’s modulus (~ 1kPa). To highlight the importance of including biomimetic interfaces into microscale models we report multi-tiered readouts from primary rat cortical cells cultured on the self-assembled membrane and compare a panel of mRNA targets with primary brain tissue signatures. We anticipate that the modular approach and simplified operational workflows presented here will enable a wide range of research groups to incorporate microfluidic barrier tissue models into their work.</p></div

LNA Flow-FISH detection of miR155 in Jurkat cells activated by PMA and Ionomycin.

<p>Jurkat cells stimulated with PMA and Ionomycin for 0, 8, 16, 20, and 24 h were loaded in duplicate into the 10-chamber chip as shown in A. The top 5 chambers were hybridized with miR155 probe, whereas the bottom 5 chambers were hybridized to scrambled probe for negative control. B. overlay of miR155 fluorescence histograms measured by LNA flow-FISH. C. miR155 median fluorescence normalized as a percentage of the maximum fluorescence, showing significant increase from 0 h at 16, 20, and 24 h stimulation. (* p<0.01) The increase at 8 h was not statistically significant (** p>0.01). D. RT-qPCR of miR155 fold changes in Jurkat cells treated with PMA and Ionomycin, also showing significant miR155 increase starting at 16 h stimulation, but not at 8 h. (** p>0.01, * p<0.01).</p

Single Cell MicroRNA Analysis Using Microfluidic Flow Cytometry

<div><p>MicroRNAs (miRNAs) are non-coding small RNAs that have cell type and cell context-dependent expression and function. To study miRNAs at single-cell resolution, we have developed a novel microfluidic approach, where flow fluorescent <em>in situ</em> hybridization (flow-FISH) using locked-nucleic acid probes is combined with rolling circle amplification to detect the presence and localization of miRNA. Furthermore, our flow cytometry approach allows analysis of gene-products potentially targeted by miRNA together with the miRNA in the same cells. We demonstrate simultaneous measurement of miR155 and CD69 in 12-O-tetradecanoylphorbol 13-acetate (PMA) and Ionomycin stimulated Jurkat cells. The flow-FISH method can be completed in ∼10 h, utilizes only 170 nL of reagent per experimental condition, and is the first to directly detect miRNA in single cells using flow cytometry.</p> </div

Microfluidic chip and LNA flow-FISH method schematic.

<p>A. 10-chamber microfluidic chip. Cells prepared in each of the holding chambers can be detached and driven to the center of the chip shown in B, for hydrodynamic focus and flow cytometry. The signal from miRNA155 hybridized to the LNA probe is amplified using RCA method depicted in C. The DIG label on both ends of the LNA probe is recognized by anti-DIG mAb, and two RCA probes (+ and -) bind to each anti-DIG mAb and are ligated to two additional oligonucleotides to form circular templates for rolling circle amplification. The resultant concatemer products can be visualized using microscopy as well as measured using flow cytometry.</p

Investigation of type-I interferon dysregulation by arenaviruses : a multidisciplinary approach.

This report provides a detailed overview of the work performed for project number 130781, 'A Systems Biology Approach to Understanding Viral Hemorrhagic Fever Pathogenesis.' We report progress in five key areas: single cell isolation devices and control systems, fluorescent cytokine and transcription factor reporters, on-chip viral infection assays, molecular virology analysis of Arenavirus nucleoprotein structure-function, and development of computational tools to predict virus-host protein interactions. Although a great deal of work remains from that begun here, we have developed several novel single cell analysis tools and knowledge of Arenavirus biology that will facilitate and inform future publications and funding proposals