Femtosecond optoinjection of intact tobacco BY-2 cells using a reconfigurable photoporation platform

A tightly-focused ultrashort pulsed laser beam incident upon a cell membrane has previously been shown to transiently increase cell membrane permeability while maintaining the viability of the cell, a technique known as photoporation. This permeability can be used to aid the passage of membrane-impermeable biologically-relevant substances such as dyes, proteins and nucleic acids into the cell. Ultrashort-pulsed lasers have proven to be indispensable for photoporating mammalian cells but they have rarely been applied to plant cells due to their larger sizes and rigid and thick cell walls, which significantly hinders the intracellular delivery of exogenous substances. Here we demonstrate and quantify femtosecond optical injection of membrane impermeable dyes into intact BY-2 tobacco plant cells growing in culture, investigating both optical and biological parameters. Specifically, we show that the long axial extent of a propagation invariant (“diffraction-free”) Bessel beam, which relaxes the requirements for tight focusing on the cell membrane, outperforms a standard Gaussian photoporation beam, achieving up to 70% optoinjection efficiency. Studies on the osmotic effects of culture media show that a hypertonic extracellular medium was found to be necessary to reduce turgor pressure and facilitate molecular entry into the cells.Publisher PDFPeer reviewe

Dynamics of primary and secondary microbubbles created by laser-induced breakdown of an optically trapped nanoparticle

Laser-induced breakdown of an optically trapped nanoparticle is a unique system for studying cavitation dynamics. It offers additional degrees of freedom, namely the nanoparticle material, its size, and the relative position between the laser focus and the center of the optically trapped nanoparticle. We quantify the spatial and temporal dynamics of the cavitation and secondary bubbles created in this system and use hydrodynamic modeling to quantify the observed dynamic shear stress of the expanding bubble. In the final stage of bubble collapse, we visualize the formation of multiple submicrometer secondary bubbles around the toroidal bubble on the substrate. We show that the pattern of the secondary bubbles typically has its circular symmetry broken along an axis whose unique angle rotates over time. This is a result of vorticity along the jet towards the boundary upon bubble collapse near solid boundaries.Y. Arita, M. Antkowiak, V. Venugopalan, F. J. Gunn-Moore and K. Dholaki

Willin/FRMD6 expression activates the Hippo signaling pathway kinases in mammals and antagonizes oncogenic YAP

The Salvador/Warts/Hippo (Hippo) signaling pathway defines a novel signaling cascade regulating cell contact inhibition, organ size control, cell growth, proliferation, apoptosis and cancer development in mammals. The Drosophila melanogaster protein Expanded acts in the Hippo signaling pathway to control organ size. Previously, willin/FRMD6 has been proposed as the human orthologue of Expanded. Willin lacks C-terminal sequences that are present in Expanded and, to date, little is known about the functional role of willin in mammalian cells. When willin is expressed in D. melanogaster epithelial tissues, it has the same subcellular localization as Expanded, but cannot rescue growth defects associated with expanded deficiency. However, we show that ectopic willin expression causes an increase in phosphorylation of the core Hippo signaling pathway components MST1/2, LATS1 and YAP, an effect that can be antagonized by ezrin. In MCF10A cells, loss of willin expression displays epithelial-to-mesenchymal transition features and willin overexpression antagonizes YAP activity via the N-terminal FERM domain of willin. Therefore, in mammalian cells willin influences Hippo signaling activity by activating the core Hippo pathway kinase cassette.L Angus, S Moleirinho, L Herron, A Sinha, X Zhang, M Niestrata, K Dholakia, MB Prystowsky, KF Harvey, PA Reynolds, and FJ Gunn-Moor

KIBRA exhibits MST-independent functional regulation of the Hippo signaling pathway in mammals

The Salvador/Warts/Hippo (Hippo) signaling pathway defines a novel signaling cascade regulating cell contact inhibition, organ size control, cell growth, proliferation, apoptosis and cancer development in mammals. The upstream regulation of this pathway has been less well defined than the core kinase cassette. KIBRA has been shown to function as an upstream member of the Hippo pathway by influencing the phosphorylation of LATS and YAP, but functional consequences of these biochemical changes have not been previously addressed. We show that in MCF10A cells, loss of KIBRA expression displays epithelial-to-mesenchymal transition (EMT) features, which are concomitant with decreased LATS and YAP phosphorylation, but not MST1/2. In addition, ectopic KIBRA expression antagonizes YAP via the serine 127 phosphorylation site and we show that KIBRA, Willin and Merlin differentially regulate genes controlled by YAP. Finally, reduced KIBRA expression in primary breast cancer specimens correlates with the recently described claudin-low subtype, an aggressive sub-group with EMT features and a poor prognosis

From Histology and Imaging Data to Models for In-Stent Restenosis

The implantation of stents has been used to treat coronary artery stenosis for several decades. Although stenting is successful in restoring the vessel lumen and is a minimally invasive approach, the long-term outcomes are often compromised by in-stent restenosis (ISR). Animal models have provided insights into the pathophysiology of ISR and are widely used to evaluate candidate drug inhibitors of ISR. Such biological models allow the response of the vessel to stent implantation to be studied without the variation of lesion characteristics encountered in patient studies.This paper describes the development of complementary in silico models employed to improve the understanding of the biological response to stenting using a porcine model of restenosis. This includes experimental quantification using microCT imaging and histology and the use of this data to establish numerical models of restenosis. Comparison of in silico results with histology is used to examine the relationship between spatial localization of fluid and solid mechanics stimuli immediately post-stenting. Multi-scale simulation methods are employed to study the evolution of neointimal growth over time and the variation in the extent of neointimal hyperplasia within the stented region. Interpretation of model results through direct comparison with the biological response contributes to more detailed understanding of the pathophysiology of ISR, and suggests the focus for follow-up studies.In conclusion we outline the challenges which remain to both complete our understanding of the mechanisms responsible for restenosis and translate these models to applications in stent design and treatment planning at both population-based and patient-specific levels

Phylogenomics and the rise of the angiosperms

Angiosperms are the cornerstone of most terrestrial ecosystems and human livelihoods1,2. A robust understanding of angiosperm evolution is required to explain their rise to ecological dominance. So far, the angiosperm tree of life has been determined primarily by means of analyses of the plastid genome3,4. Many studies have drawn on this foundational work, such as classification and first insights into angiosperm diversification since their Mesozoic origins5,6,7. However, the limited and biased sampling of both taxa and genomes undermines confidence in the tree and its implications. Here, we build the tree of life for almost 8,000 (about 60%) angiosperm genera using a standardized set of 353 nuclear genes8. This 15-fold increase in genus-level sampling relative to comparable nuclear studies9 provides a critical test of earlier results and brings notable change to key groups, especially in rosids, while substantiating many previously predicted relationships. Scaling this tree to time using 200 fossils, we discovered that early angiosperm evolution was characterized by high gene tree conflict and explosive diversification, giving rise to more than 80% of extant angiosperm orders. Steady diversification ensued through the remaining Mesozoic Era until rates resurged in the Cenozoic Era, concurrent with decreasing global temperatures and tightly linked with gene tree conflict. Taken together, our extensive sampling combined with advanced phylogenomic methods shows the deep history and full complexity in the evolution of a megadiverse clade