Publications

2018

• The Future of Multiplexed Eukaryotic Genome Engineering
  
  • Thompson David
  • Aboulhouda Soufiane
  • Hysolli Eriona
  • Smith Cory
  • Wang Stan
  • Castanon Oscar
  • Church George
  ACS Chemical Biology, American Chemical Society, 2018, 13 (2), pp.313 - 325. Multiplex genome editing is the simultaneous introduction of multiple distinct modifications to a given genome. Though in its infancy, maturation of this field will facilitate powerful new biomedical research approaches and will enable a host of far-reaching biological engineering applications, including new therapeutic modalities and industrial applications, as well as “genome writing” and de-extinction efforts. In this Perspective, we focus on multiplex editing of large eukaryotic genomes. We describe the current state of multiplexed genome editing, the current limits of our ability to multiplex edits, and provide perspective on the many applications that fully realized multiplex editing technologies would enable in higher eukaryotic genomes. We offer a broad look at future directions, covering emergent CRISPR-based technologies, advances in intracellular delivery, and new DNA assembly approaches that may enable future genome editing on a massively multiplexed scale. (10.1021/acschembio.7b00842)
  DOI : 10.1021/acschembio.7b00842
• Highly concentrated collagen solutions leading to transparent scaffolds of controlled three-dimensional organizations for corneal epithelial cell colonization
  
  • Tidu Aurelien
  • Ghoubay-Benallaoua Djida
  • Teulon Claire
  • Asnacios Sophie
  • Grieve Kate
  • Portier François
  • Schanne-Klein Marie-Claire
  • Borderie Vincent M.
  • Mosser Gervaise
  Biomaterials Science, Royal Society of Chemistry (RSC), 2018, 6 (6), pp.1492-1502. This study aimed at controlling both the organization and the transparency of dense collagen scaffolds making use of the lyotropic mesogen properties of collagen. Cholesteric or plywood-like liquid crystal phases were achieved using mixtures of acetic and hydrochloric acids as solvents. The critical pH at which the switch between the two phases occurred was around pH = 3. The use of the two acids led to fibril-lated collagen I scaffolds, whose visual aspect ranged from opaque to transparent. Rheological investigations showed that viscoelastic properties of the plywood-like solutions were optimized for molding due to faster recovery. They also confirmed the correlation between the elastic modulus and the diameter of collagen fibrils obtained after fibrillogenesis under ammonia vapor. Human corneal epithelial cells, grown from donor limbal explants, were cultured both on transparent plywood-like matrices and on human amniotic membranes for 14 days. The development of corneal epithelium and the preservation of epi-thelial stem cells were checked by optical microscopy, colony formation assay, immuno-fluorescence and quantitative polymerase chain reaction. A higher level of amplification of limbal stem cells was obtained with collagen matrices compared with amniotic membranes, showing the high biocompatibility of our scaffolds. We therefore suggest that collagen solutions presenting both plywood-like organization and transparency might be of interest for biomedical applications in ophthalmology. (10.1039/c7bm01163f)
  DOI : 10.1039/c7bm01163f
• Multiscale conformational dynamics probed by time-resolved circular dichroism.
  
  • Schmid Marco
  • Changenet Pascale
  • Hache François
  Proceedings of SPIE, the International Society for Optical Engineering, SPIE, The International Society for Optical Engineering, 2018.
• Dual-color deep-tissue three-photon microscopy with a multiband infrared laser
  
  • Guesmi Khmaies
  • Abdeladim Lamiae
  • Tozer Samuel
  • Mahou Pierre
  • Kumamoto Takuma
  • Jurkus Karolis
  • Rigaud Philippe
  • Loulier Karine
  • Dray Nicolas
  • Georges Patrick
  • Hanna Marc
  • Livet Jean
  • Supatto Willy
  • Beaurepaire Emmanuel
  • Druon Frédéric
  Light: Science and Applications, Nature Publishing Group, 2018, 7 (1), pp.12. Multiphoton microscopy combined with genetically encoded fluorescent indicators is a central tool in biology. Three-photon (3P) microscopy with excitation in the short-wavelength infrared (SWIR) water transparency bands at 1.3 and 1.7 µm opens up new opportunities for deep-tissue imaging. However, novel strategies are needed to enable in-depth multicolor fluorescence imaging and fully develop such an imaging approach. Here, we report on a novel multiband SWIR source that simultaneously emits ultrashort pulses at 1.3 and 1.7 µm that has characteristics optimized for 3P microscopy: sub-70 fs duration, 1.25 MHz repetition rate, and µJ-range pulse energy. In turn, we achieve simultaneous 3P excitation of green fluorescent protein (GFP) and red fluorescent proteins (mRFP, mCherry, tdTomato) along with third-harmonic generation. We demonstrate in-depth dual-color 3P imaging in a fixed mouse brain, chick embryo spinal cord, and live adult zebrafish brain, with an improved signal-to-background ratio compared to multicolor two-photon imaging. This development opens the way towards multiparametric imaging deep within scattering tissues. (10.1038/s41377-018-0012-2)
  DOI : 10.1038/s41377-018-0012-2
• A mechanism for CO regulation of ion channels
  
  • Kapetanaki Sofia M.
  • Burton Mark J.
  • Basran Jaswir
  • Uragami Chiasa
  • Moody Peter C. E.
  • Mitcheson John S.
  • Schmid Ralf
  • Davies Noel W.
  • Dorlet Pierre
  • Vos Marten H.
  • Storey Nina M.
  • Raven Emma
  Nature Communications, Nature Publishing Group, 2018, 9, pp.907. Despite being highly toxic, carbon monoxide (CO) is also an essential intracellular signalling molecule. The mechanisms of CO-dependent cell signalling are poorly defined, but are likely to involve interactions with heme proteins. One such role for CO is in ion channel regulation. Here, we examine the interaction of CO with K$_{ATP}$ channels. We find that CO activates K$_{ATP}$ channels and that heme binding to a CXXHX$_{16}$H motif on the SUR2A receptor is required for the CO-dependent increase in channel activity. Spectroscopic and kinetic data were used to quantify the interaction of CO with the ferrous heme-SUR2A complex. The results are significant because they directly connect CO-dependent regulation to a heme-binding event on the channel. We use this information to present molecular-level insight into the dynamic processes that control the interactions of CO with a heme-regulated channel protein, and we present a structural framework for understanding the complex interplay between heme and CO in ion channel regulation. (10.1038/s41467-018-03291-z)
  DOI : 10.1038/s41467-018-03291-z