Macromolecular cryoprotectants for the preservation of mammalian cell culture: lessons from crowding, overview and perspectives

Cryopreservation is a process used for the storage of mammalian cells at a very low temperature, in a state of ‘suspended animation’.

Evolution of diapause in the African turquoise killifish by remodeling ancient gene regulatory landscape

Suspended animation states such as hibernation or diapause allow organisms to survive extreme environments. But the mechanisms underlying the evolution of these extreme survival states are unknown. The African turquoise killifish has evolved diapause as a form of suspended development to survive the complete drought that occurs every year in its habitat. Here we show that many gene duplicates - paralogs - exhibit specialized expression in diapause versus normal development in the African turquoise killifish. Surprisingly, paralogs with specialized expression in diapause are evolutionarily very ancient, and they are also present even in vertebrates that do not exhibit diapause. Profiling the chromatin accessibility landscape among different fish species reveals an evolutionarily recent increase in chromatin accessibility at these very ancient paralogs, suggesting rewiring of their regulatory landscape. The increase in chromatin accessibility in the African turquoise killifish is linked to the presence of new binding sites for transcription factors (e.g., FOXO, REST, and PPAR), due to both de novo mutations and transposable element insertion. Interestingly, accessible chromatin regions in diapause are enriched for lipid metabolism genes. By performing lipidomics in different fish species, we uncover a specific lipid profile in African turquoise killifish embryos in diapause. Notably, select very long-chain fatty acids are high in diapause, suggesting they may be used for long-term survival in this state. Together, our multi-omic analysis indicates that diapause is driven by regulatory innovation of very ancient gene programs that are critical for survival. Our work also suggests a mechanism for how complex adaptations evolve in nature and offers strategies by which a suspended animation program could be reactivated in other species for long-term preservation.

Bacterial small RNAs and host epigenetic effectors of a transgenerational memory of pathogens in C. elegans.

The inheritance of memories is adaptive for survival. Microbes interact with all organisms challenging their immunity and triggering behavioral adaptations. Some of these behaviors induced by bacteria can be inherited although the mechanisms of action are largely unexplored. In this work, we use C. elegans and its bacteria to study the transgenerational RNA dynamics of an interspecies crosstalk leading to a heritable behavior. Heritable responses to bacterial pathogens in the nematode include avoidance and pathogen-induced diapause (PIDF), a state of suspended animation to evade the pathogen threat. We identify a small RNA RsmY, involved in quorum sensing from P. aeruginosa as required for initiation of PIDF. Histone methyltransferase SET-18/SMYD3 is also needed for PIDF initiation in C. elegans. In contrast, SET-25/EHMT2 is necessary for the maintenance of the memory of pathogen exposure in the transgenerational lineage. This work can be a starting point to understanding microbiome-induced inheritance of acquired traits.

Development of the African Killifish as a New Model to Study Aging and Suspended animation

We have pioneered a new model organism for aging research, the naturally short-lived African killifish Nothobranchius furzeri. The African killifish lives in ephemeral pools of water in Africa, and has evolved a short life cycle adapted to this habitat. Its embryos can also resist drought until the next wet season in a state of ‘suspended life’. In laboratory conditions, the African killifish has a maximal lifespan of about 4-6 months, and is, so far, the shortest-lived vertebrate that can be bred in captivity. We have successfully transformed this natural short-lived vertebrate into a usable model organism for aging research, including de novo assembly of the genome and CRISPR-Cas9 mediated genome-editing. Our goal is to use this model to discover new principles underlying aging, longevity, and ‘suspended animation’ in vertebrates.