Biology subjects

Megaly, M.

Publications and source records attributed to Megaly, M..

3 recordsLinked to original sources

bioRxiv2024

Vitrification and rapid rewarming of precision-cut liver slices for pharmacological and biomedical research

Background and AimsHigh-throughput in vitro pharmacological toxicity testing is essential for drug discovery. Precision-cut liver slices (PCLS) provide a robust system for screening that is more representative of the complex 3D structure of the whole liver than isolated hepatocytes. However, PCLS are not available as off-the-shelf products, significantly limiting their translational potential. Cryopreservation could solve this bottleneck by effectively preserving PCLS indefinitely until their time of use. Conventional cryopreservation (slow cooling in DMSO-forming ice) results in poor PCLS viability and function and, therefore, has proven unsuitable. Here, we explore an "ice-free" cryopreservation approach called vitrification and focus on culturing and assessing PCLS for 3 days post-vitrification and rewarming, given that most acute drug toxicity tests are conducted over 24h. MethodsRat liver slices were diffusively loaded with a cryoprotective agent (CPA) cocktail consisting of EG and Sucrose. The CPA-loaded PCLS were placed on a polymer cryomesh, vitrified in liquid nitrogen (LN2), and rapidly rewarmed in CPA. The vitrified and rewarmed PCLS were subsequently cultured in a controlled volume of serum-free, chemically defined media for 3 days. ResultsThe cryopreserved PCLS maintained high viability, morphology, function, enzymatic activity, and drug toxicity response. Results show that the vitrified PCLS perform comparably to untreated controls and significantly outperform conventionally cryopreserved PCLS in all assessments (p < 0.05). ConclusionsRapid vitrification and rewarming of PCLS using cryomesh enabled successful preservation and culture. This approach maintained high viability, function, enzymatic activity, and drug response for 3 days in culture, similar to controls. Impact and ImplicationsThe implications of using vitrification to store PCLS are extensive. This technology provides an exciting opportunity for the development of an "off-the-shelf" cold supply chain of human PCLS from organs declined for transplant, which are sliced, cryopreserved, and stored in a repository and available for on-demand shipping for industrial and academic biomedical research. This would also allow PCLS to become a scalable, reproducible, wide-ranging, and population-representative source of tissue that can accurately mimic in-vivo conditions of the human liver. These transformative technologies could revolutionize our practice in studying not just the metabolism of drugs but also increase our capacity to study the zonal progression of many liver diseases and conduct other exciting biomedical research.

bioengineering↗

bioRxiv2023

Notch signalling plays a critical role in patterning the ventral mesoderm during early embryogenesis in Drosophila melanogaster

Notch signalling is a critical regulator of multiple developmental processes through its ability to control gene expression and thereby influence cell fate specification and cell proliferation through direct cell-cell communication. Although Notch signalling has been implicated in myogenesis during late embryogenesis, its role in early mesoderm development has been largely unexplored. Endocytosis of the Notch ligand Delta and the Notch receptor extracellular domain, a critical step in Notch pathway activation, has been extensively observed in the ventral mesoderm of the early Drosophila embryo, indicating a potential for Notch signalling activity in this early germ layer. Here we present evidence that genes critical to mesoderm development require and are responsive to Notch signalling activity. Using a novel light-inducible Optogenetic variant of the Notch intracellular domain (OptoNotch), which affords precise spatial and temporal control over ectopic activation of Notch signalling, in combination with high-resolution fluorescent RNA in situ hybridization and qPCR, we identified a set of mesodermal genes whose expression is directly regulated by Notch signalling. We also provide evidence that Notch signalling indirectly regulates the dorsal-ventral patterning program mediated by the Toll signalling pathway through the Dorsal/ Twist/ Snail gene network. Our findings demonstrate that Notch signalling regulates ventral mesoderm patterning and is critical for establishing the mesoderm-mesectoderm-ectoderm boundary by regulating gene expression patterns and providing negative feedback on the upstream patterning network.

developmental biology↗

bioRxiv2023

Notch1 forms nuclear transcriptional condensates that drive target gene expression.

The Notch receptor is a pleiotropic signaling protein that translates intercellular ligand interactions into changes in gene expression via the nuclear localization of the Notch intracellular Domain (NICD). Using a combination of immunohistochemistry, RNA in situ, Optogenetics and super-resolution live imaging of transcription in human cells, we show that the N1ICD can form condensates that positively facilitate Notch target gene expression. We determined that N1ICD undergoes Phase Separation Coupled Percolation (PSCP) into transcriptional condensates, which recruit, enrich, and encapsulate a broad set of core transcriptional proteins. We show that the capacity for condensation is due to the intrinsically disordered transcriptional activation domain of the N1ICD. In addition, the formation of such transcriptional condensates acts to promote Notch-mediated super enhancer-looping and concomitant activation of the MYC protooncogene expression. Overall, we introduce a novel mechanism of Notch1 activity in which discrete changes in nuclear N1ICD abundance are translated into the assembly of transcriptional condensates that facilitate gene expression by enriching essential transcriptional machineries at target genomic loci.

cell biology↗