Oomycetes and fungi are microorganisms whose pathogenic (invasive) growth can cause diseases that are responsible for significant ecological and economic losses. Such growth requires the generation of a protrusive force, the magnitude and direction of which involves a balance between turgor pressure and localised yielding of the cell wall and the cytoskeleton. To study invasive growth in individual hyphae we have developed a lab-on-a-chip platform with integrated force-sensors based on elastomeric polydimethylsiloxane (PDMS) micro-pillars. With this platform we are able to measure protrusive force (both magnitude and direction) and hyphal morphology. To show the usefulness of the platform, the oomycete Achlya bisexualis was inoculated and grown on a chip. Growth of individual hyphae into a micro-pillar revealed a maximum total force of 10 μN at the hyphal tip. The chips had no discernible effect on hyphal growth rates, but hyphae were slightly thinner in the channels on the chips compared to those on agar plates. When the hyphae contacted the pillars tip extension decreased while tip width increased. A. bisexualis hyphae were observed to reorient their growth direction if they were not able to bend and effectively grow over the pillars. Estimates of the pressure exerted on a pillar were 0.09 MPa, which given earlier measures of turgor of 0.65 MPa would indicate low compliance of the cell wall. The platform is adaptable to numerous cells and organisms that exhibit tip-growth. It provides a useful tool to begin to unravel the molecular mechanisms that underlie the generation of a protrusive force.

Gambogic acid (GA) has been shown to inhibit cancer cell proliferation, induce apoptosis, and enhance reactive oxygen species accumulation. However, whether GA could improve multidrug resistance through modulating autophagy has never been explored. We demonstrated that the combination of GA and cisplatin (CDDP) resulted in a stronger growth inhibition effect on A549 and NCI‐H460 cells using the MTT assay. Furthermore, treatment with GA significantly increased autophagy in these cells. More importantly, GA‐induced cell death could be largely abolished by 3‐methyladenine (3‐MA) or chloroquine (CQ) treatment, suggesting that GA‐induced cell death was dependent on autophagy. Western blot analysis showed that GA treatment suppressed the activation of Akt, mTOR, and S6. In addition, using a GA and rapamycin combination induced more cell death compared to either GA or rapamycin alone. In summary, GA may have utility as an adjunct therapy for non‐small cell lung cancer (NSCLC) patients through autophagy‐dependent cell death, even when cancer cells have developed resistance to apoptosis.

This paper describes the design, fabrication and characterisation of a novel monolithic lab-on-a-chip (LOC) platform combining the trapping and germination of individual zoospores of the oomycete Achlya bisexualis with elastomeric micropillar-based protrusive force sensing. The oomycetes are of significant interest due to their pathogenic capabilities, which can have profound ecological and economic impacts. Zoospore encystment and germination via a germ tube play a key role in their pathogenicity. Our platform enables the study of these processes at a single cell level through hydrodynamic trapping of zoospores and their individual compartmentalization via normally closed pneumatic membrane microvalves. Valve geometry was optimized and media exchange characterized during dynamic valve operations to enhance the capture-to-growth ratio. We demonstrate germination of A. bisexualis zoospores on the platform and report three distinct germination patterns. Once germinated, germ tubes grew down growth channels towards single elastomeric micropillars. Tracking of pillar movement allowed for the measurement of microNewton range protrusive forces imparted by the tips of the germ tubes. Results indicate that the forces generated by the germ tubes are smaller than those exerted by mature hyphae. Through the use of parallel traps, channels and pillars on the same device, the platform enables high-throughput screening (HTS) of zoospores and their generation of protrusive force, an essential component of their infective capability. Due to its versatility, it will also allow for the screening of naturally bioactive compounds and the development of new biocontrol strategies for oomycetes, and morphologically similar fungal infections, as an alternative to agrochemicals.

This paper reports the fabrication and application of a Lab-on-a-Chip platform containing single-elastomeric micropillars in channel constrictions, which enable the measurement of protrusive forces exerted by individual fungal hyphae. We show the device design, the fabrication process, and photoresist optimization required to adapt the microfluidic platform to relatively thin hyphae. To demonstrate the applicability of the devices, the oomycete Achlya bisexualis and the fungus Neurospora crassa were cultured on PDMS chips. Devices were combined with confocal imaging to study the interaction of A. bisexualis hyphae with the measurement pillars. The force exerted by individual hyphae of N. crassa was measured and compared with a hyphal growth rate and diameter. The platform provides a new tool to help understand the molecular processes that underlie protrusive growth and this may present new ways to tackle the pathogenic growth of these organisms and thus combat the loss of diversity that they cause. This paper is based on the conference proceedings presented at the 31st IEEE International Conference on Micro Electro Mechanical Systems (MEMS 2018), Belfast.

Pathogenic fungi and oomycetes give rise to a significant number of animal and plant diseases. While the spread of these pathogenic microorganisms is increasing globally, emerging resistance to antifungal drugs is making associated diseases more difficult to treat. High-throughput screening (HTS) and new developments in lab-on-a-chip (LOC) platforms promise to aid the discovery of urgently required new control strategies and anti-fungal/oomycete drugs. In this review, we summarize existing HTS and emergent LOC approaches in the context of infection strategies and invasive growth exhibited by these microorganisms. To aid this, we introduce key biological aspects and review existing HTS platforms based on both conventional and LOC techniques. We then provide an in-depth discussion of more specialized LOC platforms for force measurements on hyphae and to study electro- and chemotaxis in spores, approaches which have the potential to aid the discovery of alternative drug targets on future HTS platforms. Finally, we conclude with a brief discussion of the technical developments required to improve the uptake of these platforms into the general laboratory environment.

Using a bi-directional dual-flow RootChip with integrated force sensing micropillars, root growth and signalling responses to stress gradients and pathogens were studied in Arabidopsis thaliana , Nicotiana benthamiana and Solanum lycopersicum .