A portable and reconfigurable multi-organ platform for drug development with onboard microfluidic flow control.

The drug development pipeline is severely limited by a lack of reliable tools for prediction of human clinical safety and efficacy profiles for compounds at the pre-clinical stage. Here we present the design and implementation of a platform technology comprising multiple human cell-based tissue models in a portable and reconfigurable format that supports individual organ function and crosstalk for periods of up to several weeks. Organ perfusion and crosstalk are enabled by a precision flow control technology based on electromagnetic actuators embedded in an arrayed format on a microfluidic platform. We demonstrate two parallel circuits of connected airway and liver modules on a platform containing 62 electromagnetic microactuators, with precise and controlled flow rates as well as functional biological metrics over a two week time course. Technical advancements enabled by this platform include the use of non-sorptive construction materials, enhanced scalability, portability, flow control, and usability relative to conventional flow control modes (such as capillary action, pressure heads, or pneumatic air lines), and a reconfigurable and modular organ model format with common fluidic port architecture. We demonstrate stable biological function for multiple pairs of airway-liver models for periods of 2 weeks in the platform, with precise control over fluid levels, temperature, flow rate and oxygenation in order to support relevant use cases involving drug toxicity, efficacy testing, and organ-organ interaction.

The arginine finger domain of ExoT contributes to actin cytoskeleton disruption and inhibition of internalization of Pseudomonas aeruginosa by epithelial cells and macrophages.

Pseudomonas aeruginosa, an important nosocomial pathogen of humans, expresses a type III secretion system that is required for virulence. Previous studies demonstrated that the lung-virulent strain PA103 has the capacity to be either cytotoxic or invasive. Analyses of mutants suggest that PA103 delivers a negative regulator of invasion, or anti-internalization factor, to host cells via a type III secretion system. In this work we show that the type III secreted protein ExoT inhibits the internalization of PA103 by polarized epithelial cells (Madin-Darby canine kidney cells) and J774.1 macrophage-like cells. ExoS, which is closely related to ExoT but has additional ADP-ribosylating activity, can substitute for ExoT as an anti-internalization factor. ExoT contains a signature arginine finger domain found in GTPase-activating proteins. Mutation of the conserved arginine in ExoT diminished its anti-internalization activity and altered its ability to disrupt the actin cytoskeleton. Cell fractionation experiments showed that ExoT is translocated into host cells and that mutation of the arginine finger did not disrupt translocation. In a mouse model of acute pneumonia, PA103DeltaUDeltaT reached the lungs as efficiently as PA103DeltaU but showed reduced colonization of the liver. This finding suggests that the ability to resist internalization may be important for virulence in vivo.

Pili binding to asialo-GM1 on epithelial cells can mediate cytotoxicity or bacterial internalization by Pseudomonas aeruginosa.

The interaction of Pseudomonas aeruginosa type IV pili and the glycosphingolipid asialo-GM1 (aGM1) can mediate bacterial adherence to epithelial cells, but the steps subsequent to this adherence have not been elucidated. To investigate the result of the interaction of pili and aGM1, we used polarized epithelial monolayers of Madin-Darby canine kidney (MDCK) cells in culture, which contained little detectable aGM1 on their apical surface but were able to incorporate exogenous aGM1. Compared to an untreated monolayer, P. aeruginosa PA103 displayed an eightfold increase in association with and fivefold more cytotoxicity toward MDCK cells pretreated with aGM1. Cytotoxicity of either carrier-treated or aGM1-treated monolayers required the type III secreted protein ExoU. Asialo-GM1 pretreatment of MDCK monolayers likewise augmented bacterial internalization of an isogenic invasive strain approximately fourfold. These increases were not seen in monolayers treated with GM1, the sialyated form of the glycolipid, and were inhibited by treatment with an antibody to aGM1. Also, the aGM1-mediated adhesion, cytotoxicity, and internalization required intact type IV pili since nonpiliated PA103 mutants were unaffected by aGM1 pretreatment of MDCK cells. These results demonstrate that epithelial cell injury and bacterial internalization can proceed from the same adhesin-receptor interaction, and they indicate that P. aeruginosa exoproducts solely determine the steps subsequent to adhesion.

Pseudomonas aeruginosa gene products PilT and PilU are required for cytotoxicity in vitro and virulence in a mouse model of acute pneumonia.

Type IV pili of the opportunistic pathogen Pseudomonas aeruginosa mediate twitching motility and act as receptors for bacteriophage infection. They are also important bacterial adhesins, and nonpiliated mutants of P. aeruginosa have been shown to cause less epithelial cell damage in vitro and have decreased virulence in animal models. This finding raises the question as to whether the reduction in cytotoxicity and virulence of nonpiliated P. aeruginosa mutants are primarily due to defects in cell adhesion or loss of twitching motility, or both. This work describes the role of PilT and PilU, putative nucleotide-binding proteins involved in pili function, in mediating epithelial cell injury in vitro and virulence in vivo. Mutants of pilT and pilU retain surface pili but have lost twitching motility. In three different epithelial cell lines, pilT or pilU mutants of the strain PAK caused less cytotoxicity than the wild-type strain but more than isogenic, nonpiliated pilA or rpoN mutants. The pilT and pilU mutants also showed reduced association with these same epithelial cell lines compared both to the wild type, and surprisingly, to a pilA mutant. In a mouse model of acute pneumonia, the pilT and pilU mutants showed decreased colonization of the liver but not of the lung relative to the parental strain, though they exhibited no change in the ability to cause mortality. These results demonstrate that pilus function mediated by PilT and PilU is required for in vitro adherence and cytotoxicity toward epithelial cells and is important in virulence in vivo.

Novel effects on the Gonyaulax circadian system produced by the protein kinase inhibitor staurosporine.

The protein kinase inhibitor staurosporine was found to cause a dramatic increase in the free-running period (FRP) of circadian rhythms in the dinoflagellate Gonyaulax polyedra, and its effect was similar when added at different phases of the circadian cycle. Chronic exposure to staurosporine lengthened the FRP by as much as 7 h without significantly affecting the amplitude or waveform of the bioluminescence rhythm. The effect on the length of the FRP occurred only above a threshold concentration, and it lasted for a limited number of cycles that depended on the dose of the drug. The FRP lengthening was not evident until 23 to 26 h after staurosporine addition, even though the drug entered Gonyaulax cells in 1 h or less. When tested in combination with bright light pulses, staurosporine was found to enhance both light-induced phase advances and delays, indicating that the drug acts on circadian phototransduction. At concentrations that alter the FRP and the response to light pulses, staurosporine appears to act on a small number of protein kinases, attenuating the activity of two individual protein kinases without affecting overall phosphate incorporation into proteins in vitro.

Changes in the ultrastructural localization of mRNA for a circadian regulated protein.

The amount of the luciferin (substrate) binding protein (LBP) in extracts of the bioluminescent marine dinoflagellate Gonyaulax polyedra increases by about 10-fold during a 6-hour period spanning the end of the day- and the early night-phases, and then decreases by the same amount approximately 12 hours later. Previous studies have indicated that synthesis of the protein is regulated translationally. The experiments described here were undertaken to gain insight into the mechanism of this control. Evidence was obtained for the existence of mRNA-binding proteins that could have a regulatory function. In addition, circadian-related differences were observed in the total amount of mRNA as well as in the subcellular distribution of lbp mRNA.

Inhibitors of serine/threonine phosphoprotein phosphatases alter circadian properties in Gonyaulax polyedra.

Protein serine/threonine phosphatases were implicated in the regulation of circadian rhythmicity in the marine dinoflagellate Gonyaulax polyedra based on the effects of three inhibitors specific for protein phosphatases 1 and 2A (okadaic acid, calyculin A, and cantharidin). Chronic exposure to okadaic acid resulted in a significant period lengthening, as measured by the bioluminescent glow rhythm, whereas cantharidin and calyculin A caused large phase delays but no persistent effect on period. Short pulses of the phosphatase inhibitors resulted in phase delays that were greatest near subjective dawn. Unlike 6-dimethylaminopurine, a protein kinase inhibitor, okadaic acid, calyculin A, and cantharidin did not block light-induced phase shifts. The inhibitors tested also increased radiolabeled phosphate incorporation into Gonyaulax proteins in vivo and blocked protein phosphatase 1 and 2A activities in Gonyaulax extracts. This study indicates that protein dephosphorylation catalyzed by protein serine/threonine phosphatases is necessary for proper functioning of the circadian system.

An inhibitor of protein phosphorylation stops the circadian oscillator and blocks light-induced phase shifting in Gonyaulax polyedra.

The expression of circadian rhythmicity in Gonyaulax polyedra is strikingly altered by an inhibitor of protein phosphorylation. The effects of 6-dimethylaminopurine (6-DMAP), known to reversibly block cell division in many systems through inhibition of protein kinase activity, are described here for Gonyaulax. Its action appears to be exclusively tonic in nature; in cells continuously exposed, the period is lengthened in a concentration-dependent fashion. Shorter treatments at a higher concentration of 6-DMAP (5 mM) apparently stop the circadian oscillator, but reversibly so, since the rhythm resumes after drug removal with a phase delay approximately equal to the duration of the treatment. Pulses of the inhibitor are effective in causing phase delays at all times of the circadian cycle. In addition, 6-DMAP completely blocks light-induced phase advances and is effective in inhibiting many Gonyaulax protein kinases in vitro.

Identification of precursor to cytomegalovirus capsid assembly protein and evidence that processing results in loss of its carboxy-terminal end.

The 37-kilodalton (kDa) assembly protein of cytomegalovirus (strain Colburn) B capsids is shown to have a 40-kDa precursor. Pulse-chase radiolabeling experiments revealed that conversion of the precursor to the product was slow, requiring over 6 h for completion, and correlated with movement from the cytoplasmic to the nuclear fraction of Nonidet P-40-disrupted cells. Of these two proteins, only the 40-kDa precursor was synthesized in vitro from infected-cell RNA, consistent with its being the primary translation product. Amino acid sequence data obtained from CNBr-treated, high-performance liquid chromatography-purified assembly protein indicated that precursor translation begins at the first of two closely spaced potential initiation sites and that precursor maturation involves the loss of at least 32 amino acids from its carboxy-terminal end. It is also shown by immunological cross-reactivity and peptide similarity that three low-abundance B-capsid proteins (i.e., the 45-kilodalton [45K], 39K, and 38K proteins) are closely related to the assembly protein; the nature of this relatedness is discussed.