Biology subjects

Gielen, F.

Publications and source records attributed to Gielen, F..

9 recordsLinked to original sources

bioRxiv2025

Harnessing droplet microfluidics and morphology-based deep learning for the label-free study of polymicrobial-phage interactions

Evaluating the impact of bacteriophages on bacterial communities is required to assess the future utility of phage therapy. Methods able to study bacterial polycultures in the presence of phages are useful to mimic evolutionary pressures found in natural environments and recapitulate complex ecological contexts. Bacteriophages can drive rapid genetic and phenotypic changes in host cells. However, the presence of other bacteria can also impact bacterial densities and community structure, and classical methods remain lengthy and resource intensive. Here, we introduce a microdroplet-based encapsulation method in which bacterial co-cultures are imaged using Z-stack brightfield microscopy. The method relies on automated droplet imaging using a novel AI-based autofocus function, coupled with morphology-based deep learning models for accurate identification of two morphologically distinct bacterial species. We show that we can monitor the relative growth dynamics of P. aeruginosa and S. aureus growing in 11 picolitre droplets for up to 24 hours. We demonstrate quantification of growth rates, bacterial densities and lysis dynamics of the two species without the need for plating. We show that a potent lytic phage of P. aeruginosa can either fully lyse the initial P. aeruginosa population or keep its density low long-term when in the presence of S. aureus.

microbiology↗

bioRxiv2024

Thermostable in vitro transcription-translation for enzyme screening in microdroplets

BackgroundIn vitro expression involves the utilization of the transcription and translation machinery derived from the cell to produce one or more proteins of interest and has found widespread application in the optimization of gene circuits or metabolic pathways in synthetic biology but also in pharmaceutical manufacturing. Most in vitro expression systems available are active at moderate temperatures but to screen large libraries of natural or artificial genetic diversity for highly thermostable enzymes or enzyme variants, it is instrumental to enable protein synthesis at high temperatures. Moreover, given the fact that the main barrier toward the general use of in vitro expression is its high price compared with host-based recombinant expression, there is a need to develop alternative in vitro expression systems operating at high temperatures and compatible with technologies that enable ultrahigh-throughput screening in reduced volumes, such as microfluidic water-in-oil (w/o) droplets. ResultsTo this end, we produced high-expression cell-free extracts from Thermus thermophilus for in vitro translation and supplemented them with thermostable enzymatic cascades for energy regeneration and a moderately thermostable RNA polymerase for transcription, which ultimately limited the temperature of protein synthesis. The yield was comparable to other thermostable in vitro expression systems, while the preparation procedure is simpler and can be suited to different Thermus thermophilus strains. Furthermore, these extracts have enabled in vitro expression in microfluidic droplets at high temperatures for the first time. Although the composition of these extracts showed a high background in carboxyl esterase assays, {beta}-glucosidase and cellobiose hydrolase activities could be measured with minimal background. ConclusionsCell-free extracts from Thermus thermophilus represent a simpler alternative to heavily optimized or pure component thermostable in vitro expression systems. Moreover, due to their compatibility with droplet microfluidics and enzyme assays at high temperatures, the reported system represents a convenient gateway for enzyme screening at higher temperatures with ultrahigh-throughput.

microbiology↗

bioRxiv2023

Investigating bacteria-phage interaction dynamics using droplet-based technology

An alarming rise in antimicrobial resistance worldwide has spurred efforts into the search for alternatives to antibiotic treatments. The use of bacteriophages, bacterial viruses harmless to humans, represents a promising approach with potential to treat bacterial infections (phage therapy). Recent advances in microscopy-based single-cell techniques have allowed researchers to develop new quantitative approaches for assessing the interactions between bacteria and phages, especially the ability of phages to eradicate bacterial pathogen populations. Here we combine droplet microfluidics with fluorescence time-lapse microscopy to characterize the growth and lysis dynamics of the bacterium Escherichia coli confined in droplets when challenged with phage. We investigated phages that promote lysis of infected E. coli cells, specifically, a phage species with DNA genome, T7 (Escherichia virus T7) and two phage species with RNA genomes, MS2 (Emesvirus zinderi) and Q{beta} (Qubevirus durum). Our microfluidic trapping device generated and immobilized picoliter-sized droplets, enabling stable imaging of bacterial growth and lysis in a temperature-controlled setup. Temporal information on bacterial population size was recorded for up to 25 hours, allowing us to determine growth rates of bacterial populations helping us uncover the extent and speed of phage infection. In the long-term, the development of novel microfluidic and single-cell techniques will expedite research towards understanding the genetic and molecular basis of rapid phage-induced lysis, preempting bacterial resistance to phages and ultimately identifying key factors influencing the success of phage therapy.

microbiology↗

bioRxiv2023

Label-free analysis of bacterial growth and lysis at the single-cell level using droplet microfluidics and object detection-oriented deep learning

Bacteria identification and counting at the small population scale is important to many applications in the food safety industry, the diagnostics of infectious diseases and the study and discovery of novel antimicrobial compounds. There is still a lack of easy to implement, fast and accurate methods to count populations of motile cells at the single-cell level. Here, we report a label-free method to count and localize bacterial cells freely swimming in microfluidic anchored picolitre droplets. We used the object detection oriented YOLOv4 deep learning framework for cell detection from bright-field images obtained with an automated Z-stack setup. The neural network was trained to recognize Escherichia coli cell morphology with an average precision of approximately 84%. This allowed us to accurately identify individual cell division events, enabling the study of stochastic bacterial growth starting from initial populations as low as one cell. This work also demonstrates the ability to study single cell lysis in the presence of T7 lytic bacterial viruses (phages). The high precision in cell numbers facilitated the visualization of bacteria-phage interactions over timescale of hours, paving the way towards deciphering phage life cycles in confined environments.

microbiology↗

bioRxiv2022

High throughput steady-state enzyme kinetics measured in a parallel droplet generation and absorbance detection platform

Microfluidic water-in-oil emulsion droplets are becoming a mainstay of experimental biology, where they replace the classical test tube. In most applications (e.g. in ultrahigh throughput directed evolution) the droplet content is identical for all compartmentalized assay reactions. When emulsion droplets are used for kinetics or other functional assays, though, concentration dependencies (e.g. of initial rates for Michaelis-Menten plots) are required. Droplet-on-demand systems satisfy this need but extracting large amounts of data is challenging. Here we introduce a multiplexed droplet absorbance detector which, coupled to semi-automated droplet generation, forms a tubing-based droplet-on-demand system able to generate and extract quantitative datasets from defined concentration gradients across multiple series of droplets for multiple time points. The emergence of product is detected by reading the absorbance of the droplet sets at multiple, adjustable time points (reversing the flow direction after each detection, so that the droplets pass a line scan camera multiple times). Detection multiplexing allows absorbance values at twelve distinct positions to be measured and enzyme kinetics are recorded for label-free concentration gradients (composed of about 60 droplets each, covering as many concentrations). With a throughput of around 8640 data points per hour, a 10-fold improvement compared to the previously reported single point detection method is achieved. In a single experiment, twelve full datasets of high-resolution and high accuracy Michaelis-Menten kinetics were determined to demonstrate the potential for enzyme characterization for glycosidase substrates covering a range in enzymatic hydrolysis of seven orders of magnitude in kcat/KM. The straightforward set-up, high throughput, excellent data quality, wide dynamic range that allows coverage of diverse activities suggest that this system may serve as a miniaturized spectrophotometer to for detailed analysis of study clones emerging from large-scale combinatorial experiments. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/500969v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1e240b3org.highwire.dtl.DTLVardef@105ba2corg.highwire.dtl.DTLVardef@101cd63org.highwire.dtl.DTLVardef@1534a2e_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

Cancer-associated fibroblast-derived ROR2 induces WNT/PCP activation and polarized migration in receiving gastric cancer cells.

Bone marrow-derived mesenchymal stem cells and cancer-associated fibroblasts in the tumor-stromal environment have been linked to cancer progression in many studies. These fibroblasts provide signaling factors to the tumor cells that promote proliferation, survival, invasion, and metastasis. One signaling pathway influencing tumor cell behavior is the WNT/Planar Cell Polarity (PCP) signaling in gastric cancer. Here, we show that the gastric tumor cell line, AGS, can respond to the PCP ligand WNT5A, however, express a very low level of the bona-fide WNT/PCP receptor, ROR2. At the same time, we find that CAF display long filopodia and had significantly higher levels of ROR2 than normal gastric fibroblasts. By high-resolution imaging, we observe a direct, cytoneme-mediated transfer of a complex containing ROR2 and WNT5A from CAF to the gastric cancer cells. The amount of ROR2 transferred correlated with JNK signaling in receiving cells, showing a direct requirement for receptor transfer. Co-culture of AGS with CAF expressing a dominant-negative form of ROR2 exhibited reduced actin polarization and migration compared to wild-type CAF. Furthermore, induction of migration via paracrine ROR2 transfer was observed in a zebrafish in vivo model. These unexpected findings demonstrate a fresh role in the direct transfer of a Wnt receptor from a signal-producing cell to a receiving cell and explain the mechanism by which gastric cancer cells expressing low levels of ROR2 can respond to a WNT5A-high tumor microenvironment.

cancer biology↗

bioRxiv2022

A unified in vitro to in vivo fluorescence lifetime screening platform yields amyloid β aggregation inhibitors

Inhibiting the aggregation of amyloid {beta} (1-42) is a promising strategy for the development of disease-modifying Alzheimers disease therapeutics. To date, however, no sufficiently efficacious inhibitors have been identified, despite the best efforts of >200 advanced drug development campaigns. This failure can be attributed to limitations in current compound screening and in vivo validation assays. Here, we report an in vitro to in vivo screening platform based on the use of a fluorescence lifetime aggregation sensor. The microfluidic "nanoFLIM" assay developed circumvents issues that plague conventional assays, such as lack of reproducibility, high cost and artefactual false read-outs. The fluorescence lifetime sensor can also dynamically monitor peptide aggregation in cellular and Caenorhabditis elegans disease models, providing directly comparable aggregation kinetics, which is not achievable by any other method. The power of this unified system for accelerating hit-to-lead strategies, lowering attrition rates and expediting in vivo screening, was demonstrated with a pilot screening campaign of 445 compounds, revealing a new inhibitor that can inhibit amyloid {beta} self-assembly in vitro as well as in cellular and whole organism disease models.

molecular biology↗

bioRxiv2021

Phenotyping single-cell motility in microfluidic confinement

At all scales, the movement patterns of organisms serve as dynamic read-outs of their behaviour and physiology. We devised a novel droplet microfluidics assay to encapsulate single algal microswimmers inside closed arenas, and comprehensively studied their roaming behaviour subject to a large number of environmental stimuli. We compared two model species, Chlamydomonas reinhardtii (freshwater alga, 2 cilia), and Pyramimonas octopus (marine alga, 8 cilia), and detailed their highly-stereotyped behaviours and the emergence of a trio of macroscopic swimming states (smooth-forward, quiescent, tumbling or excitable backward). Harnessing ultralong timeseries statistics, we reconstructed the species-dependent reaction network that underlies the choice of locomotor behaviour in these aneural organisms, and discovered the presence of macroscopic non-equilibrium probability fluxes in these active systems. We also revealed for the first time how microswimmer motility changes instantaneously when a chemical is added to their microhabitat, by inducing deterministic fusion between paired droplets - one containing a trapped cell, and the other, a pharmacological agent that perturbs cellular excitability. By coupling single-cell entrapment with unprecedented tracking resolution, speed and duration, our approach offers unique and potent opportunities for diagnostics, drug-screening, and for querying the genetic basis of micro-organismal behaviour.

biophysics↗

bioRxiv2020

Improved RAD51 binders through motif shuffling based on the modularity of BRC repeats

Exchanges of protein sequence modules support leaps in function unavailable through point mutations during evolution. Here we study the role of the two RAD51-interacting modules within the eight binding BRC repeats of BRCA2. We created 64 chimeric repeats by shuffling these modules and measured their binding to RAD51. We found that certain shuffled repeats were stronger than any of the natural repeats, suggesting balancing of relative properties in BRC repeats. Surprisingly, the contribution from the two modules was poorly correlated with affinities of natural repeats, with weak BRC8 repeat containing the most effective N-terminal module. The binding of the strongest chimera, BRC8-2, to RAD51 was improved by -2.44 kCal/mol compared to the strongest natural repeat, BRC4. Crystal structure of RAD51:BRC8-2 complex shows an improved interface fit and an extended {beta}-hairpin in this repeat. BRC8-2 was shown to function in human cells, preventing the formation of nuclear foci after ionizing radiation.

biochemistry↗