Current issues in mouse genome engineering

The mouse is the foremost vertebrate experimental model because its genome can be precisely and variously engineered. Now that the mouse genome has been sequenced and annotated, the task of mutating each gene is feasible, and an international cooperation is providing mutated embryonic stem cells and mice as readily available resources. Because these resources will change biomedical research, decisions about their nature will have far-reaching effects. It is therefore timely to consider topical issues for mouse genome engineering, such as the background genotype; homologous, site-specific and transpositional recombination; conditional mutagenesis; RNA-mediated interference; and functional genomics with embryonic stem cells.     

TNF-induced necroptosis and PARP-1-mediated necrosis represent distinct routes to programmed necrotic cell death

Programmed necrosis is important in many (patho)physiological settings. For specific therapeutic intervention, however, a better knowledge is required whether necrosis occurs through one single “core program” or through several independent pathways. Previously, the poly(ADP-ribose) polymerase (PARP) pathway has been suggested as a crucial element of tumor necrosis factor (TNF)-mediated necroptosis. Here, we show that TNF-induced necroptosis and the PARP pathway represent distinct and independent routes to programmed necrosis. First, DNA-alkylating agents such as 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) or methyl methanesulfonate rapidly activate the PARP pathway, whereas this is a late and secondary event in TNF-induced necroptosis. Second, inhibition of the PARP pathway does not protect against TNF-induced necroptosis, e.g., the PARP-1 inhibitor 3-AB prevented MNNG- but not TNF-induced adenosine-5′-triposphate depletion, translocation of apoptosis-inducing factor, and necrosis. Likewise, olaparib, a more potent and selective PARP-1 inhibitor failed to block TNF-induced necroptosis, identical to knockdown/knockout of PARP-1, pharmacologic and genetic interference with c-Jun N-terminal kinases and calpain/cathepsin proteases as further components of the PARP pathway. Third, interruption of TNF-induced necroptosis by interference with ceramide generation, RIP1 or RIP3 function or by the radical scavenger butylated hydroxyanisole did not prevent programmed necrosis through the PARP pathway. In summary, our results suggest that the currently established role of the PARP pathway in TNF-induced necroptosis needs to be revised, with consequences for the design of future therapeutic strategies.     

Hydrogen effect on the mechanical behaviour and microstructural features of a Fe-Mn-C twinning induced plasticity steel

The influences of hydrogen on the mechanical properties and the fracture behaviour of Fe-22Mn-0.6C twinning induced plasticity steel have been investigated by slow strain rate tests and fractographic analysis.The steel showed high susceptibility to hydrogen embrittlement,which led to 62.9%and 74.2%reduction in engineering strain with 3.1 and 14.4 ppm diffusive hydrogen,respectively.The fracture surfaces revealed a transition from ductile to brittle dominated fracture modes with the rising hydrogen contents.The underlying deformation and fracture mechanisms were further exploited by examining the hydrogen effects on the dislocation substructure,stacking fault probability,and twinning behaviour in pre-strained slow strain rate test specimens and notched tensile specimens using coupled electron channelling contrast imaging and electron backscatter diffraction techniques.The results reveal that the addition of hydrogen promotes planar dislocation structures,earlier nucleation of stacking faults,and deformation twinning within those grains which have tensile axis orientations close to<111>//rolling direction and<112>//rolling direction.The developed twin lamellae result in strain localization and micro-voids at grain boundaries and eventually lead to grain boundary decohesion.     

Low-energy control of electrical turbulence in the heart

Controlling the complex spatio-temporal dynamics underlying life-threatening cardiac arrhythmias such as fibrillation is extremely difficult, because of the nonlinear interaction of excitation waves in a heterogeneous anatomical substrate. In the absence of a better strategy, strong, globally resetting electrical shocks remain the only reliable treatment for cardiac fibrillation. Here we establish the relationship between the response of the tissue to an electric field and the spatial distribution of heterogeneities in the scale-free coronary vascular structure. We show that in response to a pulsed electric field, E, these heterogeneities serve as nucleation sites for the generation of intramural electrical waves with a source density ρ(E) and a characteristic time, τ, for tissue depolarization that obeys the power law τ?∝?E(α). These intramural wave sources permit targeting of electrical turbulence near the cores of the vortices of electrical activity that drive complex fibrillatory dynamics. We show in vitro that simultaneous and direct access to multiple vortex cores results in rapid synchronization of cardiac tissue and therefore, efficient termination of fibrillation. Using this control strategy, we demonstrate low-energy termination of fibrillation in vivo. Our results give new insights into the mechanisms and dynamics underlying the control of spatio-temporal chaos in heterogeneous excitable media and provide new research perspectives towards alternative, life-saving low-energy defibrillation techniques.     

Distributed biological computation with multicellular engineered networks

Ongoing efforts within synthetic and systems biology have been directed towards the building of artificial computational devices using engineered biological units as basic building blocks. Such efforts, inspired in the standard design of electronic circuits, are limited by the difficulties arising from wiring the basic computational units (logic gates) through the appropriate connections, each one to be implemented by a different molecule. Here, we show that there is a logically different form of implementing complex Boolean logic computations that reduces wiring constraints thanks to a redundant distribution of the desired output among engineered cells. A practical implementation is presented using a library of engineered yeast cells, which can be combined in multiple ways. Each construct defines a logic function and combining cells and their connections allow building more complex synthetic devices. As a proof of principle, we have implemented many logic functions by using just a few engineered cells. Of note, small modifications and combination of those cells allowed for implementing more complex circuits such as a multiplexer or a 1-bit adder with carry, showing the great potential for re-utilization of small parts of the circuit. Our results support the approach of using cellular consortia as an efficient way of engineering complex tasks not easily solvable using single-cell implementations.     

Tuning charge transport in solution-sheared organic semiconductors using lattice strain

Circuits based on organic semiconductors are being actively explored for flexible, transparent and low-cost electronic applications. But to realize such applications, the charge carrier mobilities of solution-processed organic semiconductors must be improved. For inorganic semiconductors, a general method of increasing charge carrier mobility is to introduce strain within the crystal lattice. Here we describe a solution-processing technique for organic semiconductors in which lattice strain is used to increase charge carrier mobilities by introducing greater electron orbital overlap between the component molecules. For organic semiconductors, the spacing between cofacially stacked, conjugated backbones (the π-π stacking distance) greatly influences electron orbital overlap and therefore mobility. Using our method to incrementally introduce lattice strain, we alter the π-π stacking distance of 6,13-bis(triisopropylsilylethynyl) pentacene (TIPS-pentacene) from 3.33?? to 3.08??. We believe that 3.08?? is the shortest π-π stacking distance that has been achieved in an organic semiconductor crystal lattice (although a π-π distance of 3.04?? has been achieved through intramolecular bonding). The positive charge carrier (hole) mobility in TIPS-pentacene transistors increased from 0.8?cm(2)?V(-1)?s(-1) for unstrained films to a high mobility of 4.6?cm(2)?V(-1)?s(-1) for a strained film. Using solution processing to modify molecular packing through lattice strain should aid the development of high-performance, low-cost organic semiconducting devices.     

Solutions for a cultivated planet

Increasing population and consumption are placing unprecedented demands on agriculture and natural resources. Today, approximately a billion people are chronically malnourished while our agricultural systems are concurrently degrading land, water, biodiversity and climate on a global scale. To meet the world's future food security and sustainability needs, food production must grow substantially while, at the same time, agriculture's environmental footprint must shrink dramatically. Here we analyse solutions to this dilemma, showing that tremendous progress could be made by halting agricultural expansion, closing 'yield gaps' on underperforming lands, increasing cropping efficiency, shifting diets and reducing waste. Together, these strategies could double food production while greatly reducing the environmental impacts of agriculture.     

Membrane protein sequestering by ionic protein-lipid interactions

Neuronal exocytosis is catalysed by the SNAP receptor protein syntaxin-1A, which is clustered in the plasma membrane at sites where synaptic vesicles undergo exocytosis. However, how syntaxin-1A is sequestered is unknown. Here we show that syntaxin clustering is mediated by electrostatic interactions with the strongly anionic lipid phosphatidylinositol-4,5-bisphosphate (PIP2). Using super-resolution stimulated-emission depletion microscopy on the plasma membranes of PC12 cells, we found that PIP2 is the dominant inner-leaflet lipid in microdomains about 73 nanometres in size. This high accumulation of PIP2 was required for syntaxin-1A sequestering, as destruction of PIP2 by the phosphatase synaptojanin-1 reduced syntaxin-1A clustering. Furthermore, co-reconstitution of PIP2 and the carboxy-terminal part of syntaxin-1A in artificial giant unilamellar vesicles resulted in segregation of PIP2 and syntaxin-1A into distinct domains even when cholesterol was absent. Our results demonstrate that electrostatic protein-lipid interactions can result in the formation of microdomains independently of cholesterol or lipid phases.