Biology subjects

Omwenga, S.

Publications and source records attributed to Omwenga, S..

4 recordsLinked to original sources

bioRxiv2024

Impact of flagellated and elongated morphological phenotypes on the focusing behaviours of biological cells in inertial microfluidic devices

Inertial microfluidics has demonstrated tremendous potential to impact biological - and notably medical - fields, by offering a highly versatile, portable and cost-effective approach to cell focusing and sorting. While the range of applications of inertial devices spans medical diagnostics, bioprocessing or water engineering to mention a few, translation is still impeded by the lack of clear understanding of cell interactions in such devices. This often leads to bespoke designs that take years of development and characterisation for one targeted application, and limited tools for informed optimisation. A more fundamental knowledge of inertial behaviours is key to future translational works and impact, by enabling a deeper understanding of inertial forces in biological systems. Towards this goal, this paper focuses on high-throughput morphological phenotyping of the single-celled, flagellated parasite Leishmania mexicana to better understand how variations in cell body length, width and flagellated status impact the focusing patterns of highly non-spherical cells in curved inertial devices. Some of the key findings in this study include i) not all organelles, such as flagella, will alter focusing if the body shape is conserved, ii) the impact of cell shape is specific to a channel design and slight changes in e.g., cell confinement can completely change focusing patterns, iii) elongated prolate-like cells align in different orientations depending on their lateral position with a curved channel and iv) despite variabilities observed in focusing patterns for elongated versus rounder cell phenotypes, large morphological variations can be completely overcome at high Reynolds numbers so that all phenotypes tightly focus at a single position (here towards the channel outer wall). This last finding, in particular, may open new avenues for highly efficient cell enrichment processes, such as for the detection of pathogens in water.

bioengineering↗

bioRxiv2024

GREMLIN1 disrupts intestinal epithelial-mesenchymal crosstalk to induce a wnt-dependent ectopic stem cell niche via stromal remodelling

In homeostasis, counterbalanced morphogen signalling gradients along the vertical axis of the intestinal mucosa regulate the fate and function of epithelial and stromal cell compartments. Here, we used a disease-positioned mouse, and human tissue, to explore the consequences of pathological Bone Morphogenetic Protein (BMP) signalling dysregulation on epithelial- mesenchymal interaction. Aberrant pan-epithelial expression of the secreted BMP antagonist GREM1, resulted in ectopic crypt formation with lineage tracing demonstrating the presence of Lgr5(-) stem/progenitor cells. Isolated epithelial cell Grem1 expression had no effect on individual cell fate, indicating an intercompartmental impact of mucosal-wide BMP antagonism. Treatment with a novel anti-Grem1 antibody abrogated the polyposis phenotype, and triangulation of specific pathway inhibitors defined a pathological sequence of events, with wnt-ligand dependent ectopic stem cell niches formed through stromal remodelling following BMP disruption. These data support an emerging co-evolutionary model of intestinal cell compartmentalisation based on bidirectional regulation of epithelial-mesenchymal cell fate and function. One Sentence SummaryPathological epithelial GREM1 expression induces therapeutically reversible ectopic stem cell niches through stromal remodelling

cancer biology↗

bioRxiv2023

The use of imaging flow cytometry for rapid, high-throughput and automated analysis of the Leishmania mexicana promastigote cell cycle provides new insights into cell cycle events of short duration

Promastigote Leishmania mexicana have a complex cell division cycle characterised by the ordered replication of several single-copy organelles, a prolonged S phase and rapid G2 and cytokinesis phases, accompanied by cell cycle stage-associated morphological changes. Here we exploit these morphological changes to develop a high-throughput and semi-automated imaging flow cytometry (IFC) pipeline to analyse the cell cycle of L. mexicana in live cells. Firstly, we demonstrate that, unlike several other DNA stains, Vybrant DyeCycle Orange (DCO) is non-toxic and enables quantitative DNA imaging in live L. mexicana promastigotes. Secondly, by tagging the orphan spindle kinesin, KINF, with mNeonGreen, we describe KINFs cell cycle-dependent expression and localisation. Then, by combining manual gating of DCO DNA intensity profiles with automated masking and morphological measurements of parasite images, visual determination of the number of flagella per cell, and automated masking and analysis of mNG:KINF fluorescence, we provide a newly detailed description of L. mexicana promastigote cell cycle events that, for the first time, includes the durations of individual G2, mitosis and post-mitosis phases, and identifies G1 cells within the first 12 minutes of the new cell cycle. By applying IFC in this way, we were able, in minutes, to capture tens of thousands of high-quality brightfield and fluorescent images of live L. mexicana cells in solution, and to acquire quantitative data across multiple parameters for every image captured. Our custom-developed masking and gating scheme allowed us to identify elusive G2 cells and to demonstrate that the CDK-inhibitor, flavopiridol, arrests cells in G2 phase, rather than mitosis, providing proof-of-principle of the utility of IFC for drug mechanism-of-action studies. Further, the high-throughput nature of IFC allowed the close examination of promastigote cytokinesis, revealing considerable flexibility in both the timing of cytokinesis initiation and the direction of furrowing, in contrast to the related kinetoplastid parasite, Trypanosoma brucei. Significantly, our analysis demonstrate that the cleavage furrow can ingress unidirectionally from either pole of the cell, bidirectionally from both simultaneously or even commence internally along the anterior-posterior (A-P) axis. Our new pipeline offers many advantages over traditional methods of cell cycle analysis such as fluorescence microscopy and flow cytometry and paves the way for novel high-throughput analysis of Leishmania cell division. Author SummaryLeishmania mexicana is a single-celled parasite that is spread by sand flies and causes a spectrum of diseases called the leishmaniases in humans and animals. To cause disease, L. mexicana parasites must replicate and divide, and their cell division cycle has unusual and/or complex features, including that the parasite changes shape as it replicates. To aid analysis of the L. mexicana cell cycle, we developed a new quantitative DNA staining technique and also generated a fluorescent parasite cell line that highlighted when cells were dividing their DNA (mitosis) after replicating it. We then applied a high-throughput technique called imaging flow cytometry to capture images of tens of thousands of these parasites in just a few minutes. For each image, we were able to extract data about DNA replication, cell shape, whether the cells were in mitosis or not and how they divide. This provided new insights into how the parasites replicate and how long each stage of cell division takes as well as how the parasites split in two at the end of cell division. We were also able to use our analysis method to precisely determine the cell cycle stage at which a cell cycle inhibitor acts. More importantly, the imaging pipelines we have developed offer great advantages in terms of speed and depth over more traditional analysis techniques such as microscopy and should pave the way for increasingly detailed analyses of parasite cell biology in the future.

cell biology↗

bioRxiv2022

Molecular phenotyping of colorectal neoplasia shows dynamic and adaptive cancer stem cell population admixture

Intestinal homeostasis is underpinned by LGR5+ve crypt-base columnar stem cells (CBCs), but following injury, dedifferentiation results in the emergence of LGR5-ve regenerative stem cell populations (RSCs), characterised by fetal transcriptional profiles. Neoplasia hijacks regenerative signalling, so we assessed the distribution of CBCs and RSCs in mouse and human intestinal tumors. Using combined molecular-morphological analysis we demonstrate variable expression of stem cell markers across a range of lesions. The degree of CBC-RSC admixture was associated with both epithelial mutation and microenvironmental signalling disruption, and could be mapped across disease molecular subtypes. The CBC-RSC equilibrium was adaptive, with a dynamic response to acute selective pressure, and adaptability was associated with chemoresistance. We propose a fitness landscape model where individual tumors have equilibrated stem cell population distributions along a CBC-RSC phenotypic axis. Cellular plasticity is represented by position shift along this axis, and is influenced by cell-intrinsic, extrinsic and therapeutic selective pressures.

cancer biology↗