Dynamical systems based on the interplay of nonlinear feedback mechanisms are ubiquitous in nature [1][2][3][4][5] . Well-understood examples from photonics include mode locking 6 and a broad class of fractal optics 7 , including self-similarity 8 . In addition to the fundamental interest in such systems, fascinating technical functionalities that are difficult or even impossible to achieve with linear systems can emerge naturally from them 7 if the right control tools can be applied. Here, we demonstrate a method that exploits positive nonlocal feedback to initiate, and negative local feedback to regulate, the growth of ultrafast laser-induced metal-oxide nanostructures with unprecedented uniformity, at high speed, low cost and on non-planar or flexible surfaces. The nonlocal nature of the feedback allows us to stitch the nanostructures seamlessly, enabling coverage of indefinitely large areas with subnanometre uniformity in periodicity. We demonstrate our approach through the fabrication of titanium dioxide and tungsten oxide nanostructures, but it can also be extended to a large variety of other materials.The fabrication of nanostructures on surfaces is of paramount importance in nanotechnology and materials science 9 . There are several established techniques, including photolithography, electron-beam lithography, imprint lithography 10 and laser interference lithography 11 , as well as non-conventional approaches such as selfassembly 12 and direct laser writing 13 . These techniques require either high-cost, complex systems or offer limited flexibility. An alternative flexible and potentially very low-cost method is laserinduced periodic surface structuring (LIPSS). The first observation of LIPSS dates back to 1965 14 . However, after almost 50 years and a large body of published work that has demonstrated LIPSS on various metals, semiconductors and glasses [15][16][17][18][19] , the method has not found widespread use due to the stubborn problem of quality control 18,19 .Despite the evident role of self-assembly in the LIPSS process, uniformity and long-range order remain poor, a problem we identified as originating from the fact that the structures are initiated from multiple seed locations concurrently and independently, thereby producing an irregular pattern. Because the process is irreversible, without self-correction, these irregularities become frozen. Our solution to this relies on carefully exploiting feedback mechanisms to tightly regulate the formation of nanostructures induced by ultrashort pulses. This process can be summarized in three steps.(1) The laser beam, with a peak intensity close to the ablation threshold for titanium, is focused on a titanium surface, where it is scattered by existing nanostructures or any surface defects 15 . The interference of the scattered and incident fields leads to intensity variations in the immediate neighbourhood of the scattering point. (2) At points where the threshold intensity for ablation is exceeded, titanium reacts rapidly with O 2 from the air, form...

Engineered proteins must be phenotypically selected for function in the appropriate physiological context. Here, we present a versatile approach that allows generating panels of mammalian cells that express diversified heterologous protein libraries in the cytosol or subcellular compartments under stable conditions and in a single-variant-per-cell manner. To this end we adapt CRISPR/Cas9 editing technology to diversify targeted stretches of a protein of interest in situ. We demonstrate the utility of the approach by in situ engineering and intra-lysosome specific selection of an extremely pH-resistant long Stokes shift red fluorescent protein variant. Tailoring properties to specific conditions of cellular sub-compartments or organelles of mammalian cells can be an important asset to optimize various proteins, protein-based tools, and biosensors for distinct functions.

Abstract:We propose and demonstrate the use of short pulsed fiber lasers in surface texturing using MHz-repetition-rate, microjoule-and sub-microjoule-energy pulses. Texturing of titanium-based (Ti6Al4V) dental implant surfaces is achieved using femtosecond, picosecond and (for comparison) nanosecond pulses with the aim of controlling attachment of human cells onto the surface. Femtosecond and picosecond pulses yield similar results in the creation of micron-scale textures with greatly reduced or no thermal heat effects, whereas nanosecond pulses result in strong thermal effects. Various surface textures are created with excellent uniformity and repeatability on a desired portion of the surface. The effects of the surface texturing on the attachment and proliferation of cells are characterized under cell culture conditions. Our data indicate that picosecond-pulsed laser modification can be utilized effectively in low-cost laser surface engineering of medical implants, where different areas on the surface can be made cell-attachment friendly or hostile through the use of different patterns. 192-203 (1997) Lett. 17, 733-737 (2004). 25. S. P. S. Porto, P. A. Fleury, and T. C. Damen, "Raman spectra of TiO2, MgF2, Zn F2, FeF2, and MnF2," Phys. Rev. 154, 522-526 (1967

Protein engineering involves generating and screening large numbers of variants for desired properties. While modern DNA technology has made it easy to create protein diversity on the DNA level, the selection and validation of candidate proteins from large libraries remains a challenge. We built a screening platform that integrates high-quality fluorescence-based image analysis and robotic picking of bacterial colonies. It allows tracking each individual colony in a large population and collecting quantitative information on library composition during the protein evolution process. We demonstrate the power of the screening platform by optimizing a dim far-red-emitting fluorescent protein whose brightness increased several fold using iterative cycles of mutagenesis and platform-based screening. The resulting protein variant mCarmine is useful for imaging cells and structures within live tissue as well as for molecular tagging. Overall, the platform presented provides powerful, flexible, and low-cost instrumentation to accelerate many fluorescence-based protein optimization projects.

Shootin1 is a protein involved in neuronal polarization, and has been shown to be a key molecule for the positive/negative feedback loop for axon induction required during neuronal symmetry breaking. To better understand the molecular basis of shootin1 dynamics, we analysed the regulatory pathways and the expressional status of shootin1 gene during NGF-induced neuronal differentiation. We demonstrated that the isoform-1 and isoform-2 of shootin1 is differentially expressed during neuronal differentiation. By blocking individual downstream pathways of NGF signalling, we found that PI3K/Akt pathway plays a major role in the expression of shootin1 isoform-2. Western blot and RT-PCR results showed that the isoform-1 of shootin1 is constitutively expressed, while the isoform-2 is expressed in a manner that is strictly dependent on NGF-stimulation. Isoform-specific RT-PCR results demonstrated that the differential expression of the isoform-1 and isoform-2 of shootin1 is a consequence of alternative splicing of shootin1 pre-mRNA, in response to NGF-signalling. Collectively these findings provide the first information on the molecular mechanisms regulating the expression of shootin1 gene and represent the first example of NGF-induced alternative splicing process that has a regulatory role in neuritogenesis.