Biology subjects

Swart, L.

Publications and source records attributed to Swart, L..

3 recordsLinked to original sources

bioRxiv2023

Transwell-based microphysiological platform for high-resolution imaging of airway tissues

Transwell-based airway models have become increasingly important to study the effects of respiratory diseases and drug treatment at the air-liquid interface of the lung epithelial barrier. However, the underlying mechanisms at tissue and cell level often remain unclear, as transwell inserts feature limited live-cell imaging compatibility. Here, we report on a novel microphysiological platform for the cultivation of transwell-based lung tissues providing the possibility to alternate between air-liquid and liquid-liquid interfaces. While the air-liquid interface recapitulates physiological conditions for the lung model, the liquid-liquid interface enables live-imaging of the tissue at high spatiotemporal resolution. The plastics-based microfluidic platform enables insertion and recuperation of the transwell inserts, which allows for tissue cultivation and analysis under standardized well plate conditions. We used the device to monitor infections of Pseudomonas aeruginosa in human stem-cell-derived bronchial epithelial tissue. We continuously imaged the progression of a P. aeruginosa infection in real time at high resolution, which provided insights into bacterial spreading and invasion on the apical tissue surface, as well as insights into tissue breaching and destruction over time. The airway tissue culture system is a powerful tool to visualize and elucidate key processes of developing respiratory diseases and to facilitate drug testing and development.

bioengineering↗

bioRxiv2022

The large GTPase Sey1/atlastin mediates lipid droplet- and FadL-dependent intracellular fatty acid metabolism of Legionella pneumophila

The facultative intracellular bacterium Legionella pneumophila employs the Icm/Dot type IV secretion system (T4SS) to replicate in a unique membrane-bound compartment, the Legionella-containing vacuole (LCV). The endoplasmic reticulum (ER)-resident large fusion GTPase Sey1/atlastin promotes remodeling and expansion of LCVs, and the GTPase is also implicated in the formation of ER-derived lipid droplets (LDs). Here we show that LCVs intimately interact with palmitate-induced LDs in Dictyostelium discoideum amoeba. Comparative proteomics of LDs isolated from the D. discoideum parental strain Ax3 or {otimes}sey1 revealed 144 differentially produced proteins, of which 7 or 22 were exclusively detected in LDs isolated from strain Ax3 or {otimes}sey1, respectively. Using dually fluorescence-labeled amoeba producing the LCV marker P4C-GFP or AmtA-GFP and the LD marker mCherry-perilipin, we discovered that Sey1 and the L. pneumophila Icm/Dot T4SS as well as the effector LegG1 promote LCV-LD interactions. In vitro reconstitution of the LCV-LD interactions using purified LCVs and LDs from D. discoideum Ax3 or {otimes}sey1 revealed that Sey1 and GTP promote this process. The LCV-LD interactions were impaired for {otimes}sey1-derived LDs, suggesting that Sey1 regulates LD composition. Palmitate promoted the growth of (i) L. pneumophila wild-type in D. discoideum Ax3 but not in {otimes}sey1 mutant amoeba and (ii) L. pneumophila wild-type but not {otimes}fadL mutant bacteria lacking a homologue of the E. coli fatty acid transporter FadL. Finally, isotopologue profiling indicated that intracellular L. pneumophila metabolizes 13C-palmitate, and its catabolism was reduced in D. discoideum {otimes}sey1 and L. pneumophila {otimes}fadL. Taken together, our results reveal that Sey1 mediates LD- and FadL-dependent fatty acid metabolism of intracellular L. pneumophila.

microbiology↗

bioRxiv2022

Nanoparticle-mediated Targeting of the Fusion Gene RUNX1/ETO in t(8;21)-positive Acute Myeloid Leukaemia

A hallmark of acute myeloid leukaemias (AMLs) are chromosomal rearrangements that give rise to novel leukaemia-specific fusion genes. Most of these fusion genes are both initiating and driving events in AML and therefore constitute ideal therapeutic targets but are challenging to target by conventional drug development. siRNAs are frequently used for the specific suppression of fusion gene expression but require special formulations for efficient in vivo delivery. Here we describe the use of siRNA-loaded lipid nanoparticles for the specific therapeutic targeting of the leukaemic fusion gene RUNX1/ETO. Transient knockdown of RUNX1/ETO reduces its binding to its target genes and alters the binding of RUNX1 and its co-factor CBF{beta}. Transcriptomic changes in vivo were associated with substantially increased median survival of a t(8;21)-AML mouse model. Importantly, transient knockdown in vivo causes long-lasting inhibition of leukaemic proliferation and clonogenicity, induction of myeloid differentiation and a markedly impaired re-engraftment potential in vivo. These data strongly suggest that temporary inhibition of RUNX1/ETO results in long-term restriction of leukaemic self-renewal. Our results provide proof for the feasibility of targeting RUNX1/ETO in a pre-clinical setting and support the further development of siRNA-LNPs for the treatment of fusion gene-driven malignancies.

cancer biology↗