18 results found
Bernier L, Stan G, Junier P, et al., 2022, Spores-on-a-chip: new frontiers for spore research, Trends in Microbiology, Vol: 30, Pages: 515-518, ISSN: 0966-842X
In recent years, microfluidic technologies have become widespread in biological science. However, the suitability of this technique for understanding different aspects of spore research has hardly been considered. Herein, we review recent developments in 'spores-on-a-chip' technologies, highlighting how they could be exploited to drive new frontiers in spore research.
Masters-Clark E, Clark AJ, Stanley CE, 2022, Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level, JOVE-JOURNAL OF VISUALIZED EXPERIMENTS, ISSN: 1940-087X
Tayyrov A, Stanley CE, Azevedo S, et al., 2019, Combining microfluidics and RNA-sequencing to assess the inducible defensome of a mushroom against nematodes, BMC Genomics, Vol: 20, Pages: 1-13, ISSN: 1471-2164
BackgroundFungi are an attractive source of nutrients for predators. As part of their defense, some fungi are able to induce the production of anti-predator protein toxins in response to predation. A previous study on the interaction of the model mushroom Coprinopsis cinerea by the fungivorous nematode Aphelenchus avenae on agar plates has shown that the this fungal defense response is most pronounced in the part of the mycelium that is in direct contact with the nematode. Hence, we hypothesized that, for a comprehensive characterization of this defense response, an experimental setup that maximizes the zone of direct interaction between the fungal mycelium and the nematode, was needed.ResultsIn this study, we conducted a transcriptome analysis of C. cinerea vegetative mycelium upon challenge with A. avenae using a tailor-made microfluidic device. The device was designed such that the interaction between the fungus and the nematode was confined to a specific area and that the mycelium could be retrieved from this area for analysis. We took samples from the confrontation area after different time periods and extracted and sequenced the poly(A)+ RNA thereof. The identification of 1229 differentially expressed genes (DEGs) shows that this setup profoundly improved sensitivity over co-cultivation on agar plates where only 37 DEGs had been identified. The product of one of the most highly upregulated genes shows structural homology to bacterial pore-forming toxins, and revealed strong toxicity to various bacterivorous nematodes. In addition, bacteria associated with the fungivorous nematode A. avenae were profiled with 16S rRNA deep sequencing. Similar to the bacterivorous and plant-feeding nematodes, Proteobacteria and Bacteroidetes were the most dominant phyla in A. avenae.ConclusionsThe use of a novel experimental setup for the investigation of the defense response of a fungal mycelium to predation by fungivorous nematodes resulted in the identification of a comprehen
Ghanem N, Stanley CE, Harms H, et al., 2019, Mycelial effects on phage retention during transport in a microfluidic platform, Environmental Science and Technology (Washington), Vol: 53, Pages: 11755-11763, ISSN: 0013-936X
Phages (i.e., viruses that infect bacteria) have been considered as good tracers for the hydrological transport of colloids and (pathogenic) viruses. However, little is known about interactions of phages with (fungal) mycelia as the prevalent soil microbial biomass. Forming extensive and dense networks, mycelia provide significant surfaces for phage–hyphal interactions. Here, for the first time, we quantified the mycelial retention of phages in a microfluidic platform that allowed for defined fluid exchange around hyphae. Two common lytic tracer phages (Escherichia coli phage T4 and marine phage PSA-HS2) and two mycelia of differing surface properties (Coprinopsis cinerea and Pythium ultimum) were employed. Phage–hyphal interaction energies were approximated by the extended Derjaguin–Landau–Verwey–Overbeek (XDLVO) approach of colloidal interaction. Our data show initial hyphal retention of phages of up to ≈4 × 107 plaque-forming unit (PFU) mm–2 (≈2550 PFU mm–2 s–1) with a retention efficiency depending on the hyphal and, to a lesser extent, the phage surface properties. Experimental data were supported by XDLVO calculations, which revealed the highest attractive forces for the interaction between hydrophobic T4 phages and hydrophobic C. cinerea surfaces. Our data suggest that mycelia may be relevant for the retention of phages in the subsurface and need to be considered in subsurface phage tracer studies. Mycelia–phage interactions may further be exploited for the development of novel strategies to reduce or hinder the transport of undesirable (bio) colloidal entities in environmental filter systems.
Stöckli M, Morinaka BI, Lackner G, et al., 2019, Bacteria‐induced production of the antibacterial sesquiterpene lagopodin B in <i>Coprinopsis cinerea</i>, Molecular Microbiology, Vol: 112, Pages: 605-619, ISSN: 0950-382X
Suea-Ngam A, Howes PD, Stanley CE, et al., 2019, An Exonuclease I-Assisted Silver-Metallized Electrochemical Aptasensor for Ochratoxin A Detection, ACS Sensors, Vol: 4, Pages: 1560-1568, ISSN: 2379-3694
Denninger P, Reichelt A, Schmidt VAF, et al., 2019, Distinct RopGEFs successively drive polarization and outgrowth of root hairs, Current Biology, Vol: 29, Pages: 1854-1865.e5, ISSN: 0960-9822
Root hairs are tubular protrusions of the root epidermis that significantly enlarge the exploitable soil volume in the rhizosphere. Trichoblasts, the cell type responsible for root hair formation, switch from cell elongation to tip growth through polarization of the growth machinery to a predefined root hair initiation domain (RHID) at the plasma membrane. The emergence of this polar domain resembles the establishment of cell polarity in other eukaryotic systems [1, 2, 3]. Rho-type GTPases of plants (ROPs) are among the first molecular determinants of the RHID [4, 5], and later play a central role in polar growth . Numerous studies have elucidated mechanisms that position the RHID in the cell [7, 8, 9] or regulate ROP activity [10, 11, 12, 13, 14, 15, 16, 17, 18]. The molecular players that target ROPs to the RHID and initiate outgrowth, however, have not been identified. We dissected the timing of the growth machinery assembly in polarizing hair cells and found that positioning of molecular players and outgrowth are temporally separate processes that are each controlled by specific ROP guanine nucleotide exchange factors (GEFs). A functional analysis of trichoblast-specific GEFs revealed GEF3 to be required for normal ROP polarization and thus efficient root hair emergence, whereas GEF4 predominantly regulates subsequent tip growth. Ectopic expression of GEF3 induced the formation of spatially confined, ROP-recruiting domains in other cell types, demonstrating the role of GEF3 to serve as a membrane landmark during cell polarization.
Bui T, Harting R, BrausStromeyer SA, et al., 2019, <i>Verticillium dahliae</i>transcription factors Som1 and Vta3 control microsclerotia formation and sequential steps of plant root penetration and colonisation to induce disease, New Phytologist, Vol: 221, Pages: 2138-2159, ISSN: 0028-646X
Schmieder SS, Stanley CE, Rzepiela A, et al., 2019, Bidirectional propagation of signals and nutrients in fungal networks via specialized hyphae, Current Biology, Vol: 29, Pages: 217-228.E4, ISSN: 0960-9822
Intercellular distribution of nutrients and coordination of responses to internal and external cues via endogenous signaling molecules are hallmarks of multicellular organisms. Vegetative mycelia of multicellular fungi are syncytial networks of interconnected hyphae resulting from hyphal tip growth, branching, and fusion. Such mycelia can reach considerable dimensions and, thus, different parts can be exposed to quite different environmental conditions. Our knowledge about the mechanisms by which fungal mycelia can adjust nutrient gradients or coordinate their defense response to fungivores is scarce, in part due to limitations in technologies currently available for examining different parts of a mycelium over longer time periods at the microscopic level. Here, we combined a tailor-made microfluidic platform with time-lapse fluorescence microscopy to visualize the dynamic response of the vegetative mycelium of a basidiomycete to two different stimuli. The microfluidic platform allows simultaneous monitoring at both the colony and single-hypha level. We followed the dynamics of the distribution of a locally administered nutrient analog and the defense response to spatially confined predation by a fungivorous nematode. Although both responses of the mycelium were constrained locally, we observed long-distance propagation for both the nutrient analog and defense response in a subset of hyphae. This propagation along hyphae occurred in both acropetal and basipetal directions and, intriguingly, the direction was found to alternate every 3 hr in an individual hypha. These results suggest that multicellular fungi have, as of yet, undescribed mechanisms to coordinate the distribution of nutrients and their behavioral response upon attack by fungivores.
Stanley C, Shrivastava J, Brugman R, et al., 2018, Fabrication and use of the dual-flow-rootChip for the imaging of arabidopsis roots in asymmetric microenvironments, Bio-protocol, Vol: 8, ISSN: 2331-8325
Fabrication and Use of the Dual-Flow-RootChip for the Imaging of Arabidopsis Roots in Asymmetric Microenvironments
Stanley CE, Shrivastava J, Brugman R, et al., 2018, Dual-flow-RootChip reveals local adaptations of roots towards environmental asymmetry at the physiological and genetic levels, New Phytologist, Vol: 217, Pages: 1357-1369, ISSN: 0028-646X
Roots grow in highly dynamic and heterogeneous environments. Biological activity as well as uneven nutrient availability or localized stress factors result in diverse microenvironments. Plants adapt their root morphology in response to changing environmental conditions, yet it remains largely unknown to what extent developmental adaptations are based on systemic or cell‐autonomous responses.We present the dual‐flow‐RootChip, a microfluidic platform for asymmetric perfusion of Arabidopsis roots to investigate root–environment interactions under simulated environmental heterogeneity. Applications range from investigating physiology, root hair development and calcium signalling upon selective exposure to environmental stresses to tracing molecular uptake, performing selective drug treatments and localized inoculations with microbes.Using the dual‐flow‐RootChip, we revealed cell‐autonomous adaption of root hair development under asymmetric phosphate (Pi) perfusion, with unexpected repression in root hair growth on the side exposed to low Pi and rapid tip‐growth upregulation when Pi concentrations increased. The asymmetric root environment further resulted in an asymmetric gene expression of RSL4, a key transcriptional regulator of root hair growth.Our findings demonstrate that roots possess the capability to locally adapt to heterogeneous conditions in their environment at the physiological and transcriptional levels. Being able to generate asymmetric microenvironments for roots will help further elucidate decision‐making processes in root–environment interactions.
Stanley CE, van der Heijden MGA, 2017, Microbiome-on-a-Chip: New Frontiers in Plant–Microbiota Research, Trends in Microbiology, Vol: 25, Pages: 610-613, ISSN: 0966-842X
Stanley CE, Grossmann G, Casadevall i Solvas X, et al., 2016, Soil-on-a-Chip: microfluidic platforms for environmental organismal studies, Lab on a Chip, Vol: 16, Pages: 228-241, ISSN: 1473-0197
<p>A review of the most recent developments in so-called “Soil-on-a-Chip” microfluidic technology for environmental organismal studies, including bacteria, nematodes, fungi and plants, as well as inter-organismal interactions.</p>
Stanley CE, Stöckli M, van Swaay D, et al., 2014, Probing bacterial-fungal interactions at the single cell level., Integrative Biology: interdisciplinary approaches for molecular and cellular life sciences, Vol: 6, Pages: 935-945, ISSN: 1757-9694
Interactions between fungi and prokaryotes are abundant in many ecological systems. A wide variety of biomolecules regulate such interactions and many of them have found medicinal or biotechnological applications. However, studying a fungal-bacterial system at a cellular level is technically challenging. New microfluidic devices provided a platform for microscopic studies and for long-term, time-lapse experiments. Application of these novel tools revealed insights into the dynamic interactions between the basidiomycete Coprinopsis cinerea and the bacterium Bacillus subtilis. Direct contact was mediated by polar attachment of bacteria to only a subset of fungal hyphae suggesting a differential competence of fungal hyphae and thus differentiation of hyphae within a mycelium. The fungicidal activity of B. subtilis was monitored at a cellular level and showed a novel mode of action on fungal hyphae.
van Swaay D, Mächler J-P, Stanley C, et al., 2014, A chip-to-world connector with a built-in reservoir for simple small-volume sample injection, Lab Chip, Vol: 14, Pages: 178-181, ISSN: 1473-0197
Stanley CE, Wootton RCR, deMello AJ, 2012, Continuous and segmented flow microfluidics: applications in high-throughput chemistry and biology., Chimia (Aarau), Vol: 66, Pages: 88-98, ISSN: 0009-4293
This account highlights some of our recent activities focused on developing microfluidic technologies for application in high-throughput and high-information content chemical and biological analysis. Specifically, we discuss the use of continuous and segmented flow microfluidics for artificial membrane formation, the analysis of single cells and organisms, nanomaterial synthesis and DNA amplification via the polymerase chain reaction. In addition, we report on recent developments in small-volume detection technology that allow access to the vast amounts of chemical and biological information afforded by microfluidic systems.
Stanley CE, Elvira KS, Niu XZ, et al., 2010, A microfluidic approach for high-throughput droplet interface bilayer (DIB) formation, CHEMICAL COMMUNICATIONS, Vol: 46, Pages: 1620-1622, ISSN: 1359-7345
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Stanley CE, Clarke N, Anderson KM, et al., 2006, Anion binding inhibition of the formation of a helical organogel, Chemical Communications, Pages: 3199-3199, ISSN: 1359-7345
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