A monolithic polydimethylsiloxane platform for zoospore Capture, germination and single hypha force sensing

This paper reports a triple-layer, polydimethylsiloxane (PDMS)-based lab-on-a-chip platform combining the capture and culture of individual oomycete zoospores with integrated force sensing on germinated hyphae. The platform enables the concurrent study of cell-to-cell variability in hyphal growth and protrusive force generation. To demonstrate the applicability of the platform, individual zoospores of the oomycete Achlya bisexualis were trapped by a constriction structure, cultured on the device and the micro-Newton forces exerted by hyphae measured by tracking the deflection of elastomeric micropillars. The platform provides a new tool to help understand protrusive growth on a single cell level

Resveratrol, Acetyl-Resveratrol, and Polydatin Exhibit Antigrowth Activity against 3D Cell Aggregates of the SKOV-3 and OVCAR-8 Ovarian Cancer Cell Lines

Resveratrol has aroused significant scientific interest as it has been claimed that it exhibits a spectrum of health benefits. These include effects as an anti-inflammatory and an antitumour compound. The purpose of this study was to investigate and compare any potential antigrowth effects of resveratrol and two of its derivatives, acetyl-resveratrol and polydatin, on 3D cell aggregates of the EGFR/Her-2 positive and negative ovarian cancer cell lines SKOV-3 and OVCAR-8, respectively. Results showed that resveratrol and acetyl-resveratrol reduced cell growth in the SKOV-3 and OVCAR-8 in a dose-dependant manner. The growth reduction was mediated by the induction of apoptosis via the cleavage of poly(ADP-ribose) polymerase (PARP-1). At lower concentrations, 5 and 10 µM, resveratrol, acetyl-resveratrol, and polydatin were less effective than higher concentrations, 50 and 100 µM. In SKOV-3 line, at higher concentrations, resveratrol and polydatin significantly reduced the phosphorylation of Her-2 and EGFR and the expression of Erk. Acetyl-resveratrol, on the other hand, did not change the activation of Her-2 and EGFR. Resveratrol, acetyl-resveratrol, and polydatin suppressed the secretion of VEGF in a dose-dependant fashion. In the OVCAR-8 cell line, resveratrol and acetyl-resveratrol at 5 and 10 µM increased the activation of Erk. Above these concentrations they decreased activation. Polydatin did not produce this effect. This study demonstrates that resveratrol and its derivatives may inhibit growth of 3D cell aggregates of ovarian cancer cell lines via different signalling molecules. Resveratrol and its derivatives, therefore, warrant further in vivo evaluation to assess their potential clinical utility

Participation of annexin 1 in the response of Arabidopsis thaliana to lead exposure: potential for phytoremediation

Heavy metal pollution has become a serious public health and environmental concern. Lead (Pb) is one of the heavy metals known to bioaccumulate in plants. Phytoremediation is an emerging technology based on the ability of green plants to remove Pb from the environment in a cost-efficient and ecologically sound manner. Currently, an important research focus is to seek a better understanding of the mechanisms of Pb tolerance by plant cells, with the aim of genetically engineering plants with improved tolerance to Pb, and hence better phytoremediation capabilities in the near future. Annexin, a calcium-dependent membrane-binding protein is believed to play a role in many essential cellular processes. It has been shown that expression of annexin genes from Arabidopsis thaliana are differentially regulated in response to a variety of abiotic stresses. Thus annexins are likely be involved in the response of plants to heavy metal stress. This study aimed to obtain new insights into whether annexin 1 (AnnAt1), is involved in Pb tolerance in plant cells. Message levels of AnnAt1 were assessed in response to Pb treatments using quantitative real-time PCR. Expression results were analysed using REST 2008 and normalized against the mitosis protein YLS8. We found that Pb effect on AnnAt1 expression in plants exposed to lower Pb concentrations (25 µM, 50 µM, and 75 µM) was not significantly different from the controls. However, AnnAt1 message levels doubled (2.12-fold, S.E. range is 1.77 - 2.61, p < 0.001) in seedlings treated with 100 µM Pb, in comparison to the control plants. The relative contribution of AnnAt1 in defence against Pb stress will be discussed

Exogenous double-stranded RNA inhibits the infection physiology of rust fungi to reduce symptoms in planta

Rust fungi (Pucciniales) are a diverse group of plant pathogens in natural and agricultural systems. They pose ongoing threats to the diversity of native flora and cause annual crop yield losses. Agricultural rusts are predominantly managed with fungicides and breeding for resistance, but new control strategies are needed on non-agricultural plants and in fragile ecosystems. RNA interference (RNAi) induced by exogenous double-stranded RNA (dsRNA) has promise as a sustainable approach for managing plant-pathogenic fungi, including rust fungi. We investigated the mechanisms and impact of exogenous dsRNA on rust fungi through in vitro and whole-plant assays using two species as models, Austropuccinia psidii (the cause of myrtle rust) and Coleosporium plumeriae (the cause of frangipani rust). In vitro, dsRNA either associates externally or is internalized by urediniospores during the early stages of germination. The impact of dsRNA on rust infection architecture was examined on artificial leaf surfaces. dsRNA targeting predicted essential genes significantly reduced germination and inhibited development of infection structures, namely appressoria and penetration pegs. Exogenous dsRNA sprayed onto 1-year-old trees significantly reduced myrtle rust symptoms. Furthermore, we used comparative genomics to assess the wide-scale amenability of dsRNA to control rust fungi. We sequenced genomes of six species of rust fungi, including three new families (Araucariomyceaceae, Phragmidiaceae, and Skierkaceae) and identified key genes of the RNAi pathway across 15 species in eight families of Pucciniales. Together, these findings indicate that dsRNA targeting essential genes has potential for broad-use management of rust fungi across natural and agricultural systems

The Anti-Proliferative Effect of PI3K/mTOR and ERK Inhibition in Monolayer and Three-Dimensional Ovarian Cancer Cell Models

Most ovarian cancer patients are diagnosed with advanced stage disease, which becomes unresponsive to chemotherapeutic treatments. The PI3K/AKT/mTOR and the RAS/RAF/MEK/ERK kinase signaling pathways are attractive targets for potential therapeutic inhibitors, due to the high frequency of mutations to PTEN, PIK3CA, KRAS and BRAF in several ovarian cancer subtypes. However, monotherapies targeting one of these pathways have shown modest effects in clinical trials. This limited efficacy of the agents could be due to upregulation and increased signaling via the adjacent alternative pathway. In this study, the efficacy of combined PI3K/mTOR (BEZ235) and ERK inhibition (SCH772984) was investigated in four human ovarian cancer cell lines, grown as monolayer and three-dimensional cell aggregates. The inhibitor combination reduced cellular proliferation in a synergistic manner in OV-90 and OVCAR8 monolayers and in OV-90, OVCAR5 and SKOV3 aggregates. Sensitivity to the inhibitors was reduced in three-dimensional cell aggregates in comparison to monolayers. OV-90 cells cultured in large spheroids were sensitive to the inhibitors and displayed a robust synergistic antiproliferative response to the inhibitor combination. In contrast, OVCAR8 spheroids were resistant to the inhibitors. These findings suggest that combined PI3K/mTOR and ERK inhibition could be a useful strategy for overcoming treatment resistance in ovarian cancer and warrants further preclinical investigation. Additionally, in some cell lines the use of different three-dimensional models can influence cell line sensitivity to PI3K/mTOR and RAS/RAF/MEK/ERK pathway inhibitors

Fabrication of In-Channel High-Aspect Ratio Sensing Pillars for Protrusive Force Measurements on Fungi and Oomycetes

© 1992-2012 IEEE. 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. [2018-0090

A microfluidic gradient generator to simulate the oxygen microenvironment in cancer cell culture

© 2018 Elsevier B.V. Standard cancer lab models lack many attributes of the in-vivo cancer microenvironment. Oxygen levels for example are not commonly controlled in 2D cell-culture well plate experiments. However, low O2 (hypoxia) in particular is common in cancerous tissue due to high proliferation rates of cancer cells and inadequate vasculature. Hypoxia is also associated with cancer recurrence and drug resistance. We report a microfluidic system capable of exposing a 2D cell-culture to a dissolved oxygen gradient ranging from hypoxia (<5 mg/L) to hyperoxia (40 mg/L) for over 30 min, measurable in real-time using an integrated sensor film. The film incorporates a photostable, non-cytotoxic oxygen-sensitive fluorescent dye, which exhibits a linear response and high contrast (I0/I100 = 12) within the range of interest, was integrated onto glass substrates as a cell culture substrate. To demonstrate the applicability of the platform, Ishikawa cancer cells were cultured on the platform and exposed to linear cross-stream oxygen gradients. The platform provides a valuable tool for the culture of cancer cells in an in-vivo like microenvironment and will enable more accurate screening of new anti-cancer drugs