Dongfeng Qi
email: qidongfeng@nbu.edu.cn

Xiaohan Yu

Qingwei Zhang

Dongwoo Paeng

Xiang Han

Andong Wang

Donglin Huang

Letian Wang

Zifeng Zhang

Songyan Chen

Shixun Dai

Xiang Shen
email: shenxiang@nbu.edu.cn

Fabrication of flexible transparent Ag squareshaped mesh electrode by top-flat nanosecond laser ablation

We report a facile top-flat square nanosecond (ns) laser direct writing (LDW) ablation technique in a thin silver film substrate to fabricate square silver honeycomb structure of flexible transparent electrodes. The square silver honeycomb structures feature smooth surface morphology, excellent edge definition, mechanical stability, strong adhesion to the substrate, and favorable resistance and transparency. Due to the facile cold machining by one-step top-flat square nanosecond LDW ablation of Ag film, different thicknesses of Ag mesh electrodes (20 nm, 50 nm, 160 nm) of glossy metallic honeycomb surfaces with smooth morphology and excellent edge definition can be fabricated. In particular, this strategy enables fabrication of a high square honeycomb areal density (ablated square honeycomb to the total area) Ag mesh, substantially improving transparency (>85%) without considerably sacrificing conductivity (<23.2 Ω sq-1 unit of resistance). Consequently, the proposed metallic honeycomb structure shows compatibility with polyethylene naphthalate (PEN) flexible substrate for silver-based wearable electronic devices without any protective layer over the electrodes.

Flexible transparent conductive electrodes (FTCEs) are essential components of various electronic and optoelectronic devices, such as touch screen panels (TSPs), organic light-emitting diodes (OLEDs) and solar cells. For such transparent conductive devices, indium tin oxide (ITO) prevails in the market of transparent conducting materials. [1-5] However, due to the increasing demand for flexible and wearable electronic devices, ITO suffers from several intrinsic drawbacks, since its brittleness causes cracks upon flexible test and high material cost due to indium is the rare earth material [START_REF] Ye | Metal Nanowire Networks: The Next Generation of Transparent Conductors[END_REF][START_REF] Guo | Recent Development of Transparent Conducting Oxide-Free Flexible Thin-Film Solar Cells[END_REF][START_REF] Inganas | Organic Photovoltaics Avoiding Indium[END_REF][START_REF] Hecht | Emerging Transparent Electrodes Based on Thin Films of Carbon Nanotubes, Graphene, and Metallic Nanostructures[END_REF]. Researchers have begun to seek new flexible electrode materials such as carbon nanotubes, graphene, metal (silver, copper) nanowires and transparent metal grids [START_REF] Han | Fast Plasmonic Laser Nanowelding for a Cu-Nanowire Percolation Network for Flexible Transparent Conductors and Stretchable Electronics[END_REF][START_REF] Zhang | Transparent, conductive, and flexible carbon nanotube films and their application in organic light-emitting diodes[END_REF][START_REF] Eda | Large-area ultrathin films of reduced graphene oxide as a transparent and flexible electronic material[END_REF]. Carbon nanotubes and graphene have good flexibility can sustain tension of 100% stress stretching, but as electrode materials, the conductivity and transmittance properties of carbon nanotubes or graphite are much smaller than those of ITO materials [START_REF] Song | Highly Efficient and Bendable Organic Solar Cells with Solution-Processed Silver Nanowire Electrodes[END_REF]. The conductivity and transmittance of metal silver and copper nanowires are 1~2 orders of magnitude higher than that of carbon nanotubes, and especially for Ag nanowires, the photoelectric properties (resistance: 20 Ω sq -1 , transmittance: 90 %) are comparable to those of ITO materials [START_REF] Joe | Laser-Material Interactions for Flexible Applications[END_REF][START_REF] Lee | Flexible transparent conducting electrodes based on metal meshes for organic optoelectronic device applications: a review[END_REF]. However, the random Ag, Cu and Cu (core)-Ni (shell) nanowires network fabrication using mold, bulk heating, annealing process or fast plasmonic nano-welding method increases material waste, edge roughness and processing time [START_REF] Song | Highly Efficient and Bendable Organic Solar Cells with Solution-Processed Silver Nanowire Electrodes[END_REF][START_REF] Ye | A rapid synthesis of high aspect ratio copper nanowires for high-performance transparent conducting films[END_REF]. In addition, the performance of nanowire-based transparent electrodes largely depends on the property of nanowire such as crystallization and aspect ratio. Some methods to elongate the length of the silver nanowire and improve the crystallization of nanowires have been reported recently, such as nano-welding to enhance contact of individual nanowires and pressurization at high temperature [START_REF] Han | Fast Plasmonic Laser Nanowelding for a Cu-Nanowire Percolation Network for Flexible Transparent Conductors and Stretchable Electronics[END_REF][START_REF] Song | Superstable Transparent Conductive Cu@Cu4Ni Nanowire Elastomer Composites against Oxidation, Bending, Stretching, and Twisting for Flexible and Stretchable Optoelectronics[END_REF]. However, it makes the whole process more complicated and timeconsuming.

Laser direct writing (LDW) process can effectively fabricate grid structures in the surface of solution-processed nanoparticles (NPs) thin films [START_REF] Lee | Vacuum-Free, Maskless Patterning of Ni Electrodes by Laser Reductive Sintering of NiO Nanoparticle Ink and Its Application to Transparent Conductors[END_REF][START_REF] Hong | Nonvacuum, Maskless Fabrication of a Flexible Metal Grid Transparent Conductor by Low-Temperature Selective Laser Sintering of Nanoparticle Ink[END_REF][START_REF] Joe | Laser-Material Interactions for Flexible Applications[END_REF][START_REF] Hong | Digital selective laser methods for nanomaterials: From synthesis to processing[END_REF]. Compared with traditional processing methods, LDW process of NPs of different pitches has unique advantage for the preparation of flexible transparent electrodes. Since metal films thinner than the light absorption depth is still conductive, and metal grid fabrication methods via NPs sintering and reductive sintering have been demonstrated for transparent electrodes. In this case, utilizing metal films compatible with LDW processes is a new route for the fabrication of flexible transparent electrodes. The laser direct sintering and reduction process of solution-processed NPs, in particular, can be applied to fabricate flexible transparent devices owing to lowered thermal stress exerting on thermally vulnerable polymer substrates. [1] Recently, NPs or NPs composed of Ag, Au, Cu and Ni have been widely employed because these nano-materials can be easily maintained to grid electrodes of different pitches simply by sintering nanoparticles or reductive sintering metal oxide [START_REF] Lee | Vacuum-Free, Maskless Patterning of Ni Electrodes by Laser Reductive Sintering of NiO Nanoparticle Ink and Its Application to Transparent Conductors[END_REF][START_REF] Hong | Nonvacuum, Maskless Fabrication of a Flexible Metal Grid Transparent Conductor by Low-Temperature Selective Laser Sintering of Nanoparticle Ink[END_REF][START_REF] Nam | Highly Stable Ni-Based Flexible Transparent Conducting Panels Fabricated by Laser Digital Patterning[END_REF]. Through localized laser reductive sintering and sintering of metal oxide or metal NPs, highly conductive metal electrodes can be successfully fabricated. However, resolution of electrodes fabricated through these methods is limited due to the large and uneven size of the NPs and metal oxide [START_REF] Paeng | Characteristic time scales of coalescence of silver nanocomposite and nanoparticle films induced by continuous wave laser irradiation[END_REF]. Besides, copper and nickel can be susceptible to re-oxidation at the laser direct writing process, which causes degradation of its electrical properties [START_REF] Paeng | Characteristic time scales of coalescence of silver nanocomposite and nanoparticle films induced by continuous wave laser irradiation[END_REF][START_REF] Paeng | Laser-Induced Reductive Sintering of Nickel Oxide Nanoparticles under Ambient Conditions[END_REF][START_REF] Paeng | Laser wavelength effect on laser-induced photo-thermal sintering of silver nanoparticles[END_REF][START_REF] Qi | Time-resolved analysis of thickness-dependent dewetting and ablation of silver films upon nanosecond laser irradiation[END_REF]. In this case, another route would be utilizing deposition-processed metal films compatible with laser direct ablation process to fabricate transparent electrode [START_REF] Lee | Flexible transparent conducting electrodes based on metal meshes for organic optoelectronic device applications: a review[END_REF][START_REF] Qi | Time-resolved analysis of thickness-dependent dewetting and ablation of silver films upon nanosecond laser irradiation[END_REF]. Metal patterns on flexible substrates not only meet the high transmittance, large area and low cost performance for flexible transparent electrode, but also retain the low conductivity and the ductility of the metal film [START_REF] Paeng | Low-Cost Facile Fabrication of Flexible Transparent Copper Electrodes by Nanosecond Laser Ablation[END_REF][START_REF] Wang | Mask-Free Patterning of High-Conductivity Metal Nanowires in Open Air by Spatially Modulated Femtosecond Laser Pulses[END_REF]. Besides, the LDW ablation process of ultrathin deposition-processed metal films enabled material removal without inflicting thermal damage on the underlying flexible polymer substrates [START_REF] Paeng | Low-Cost Facile Fabrication of Flexible Transparent Copper Electrodes by Nanosecond Laser Ablation[END_REF]. As a result, metallic honeycomb arrays can be directly produced on various heat-sensitive flexible substrates. However, properties and morphologies of the electrodes are not uniform after Gaussian pulse irradiation [START_REF] Qi | Time-resolved analysis of thickness-dependent dewetting and ablation of silver films upon nanosecond laser irradiation[END_REF][START_REF] Paeng | Low-Cost Facile Fabrication of Flexible Transparent Copper Electrodes by Nanosecond Laser Ablation[END_REF]. For the Gaussian beam, the temperature gradient in the beam spot results in different thermodynamic processes in the center of the spot and the peripheral spot, which affects the quality of the metallic honeycomb transparent electrodes [START_REF] Lee | Vacuum-Free, Maskless Patterning of Ni Electrodes by Laser Reductive Sintering of NiO Nanoparticle Ink and Its Application to Transparent Conductors[END_REF][START_REF] Hong | Nonvacuum, Maskless Fabrication of a Flexible Metal Grid Transparent Conductor by Low-Temperature Selective Laser Sintering of Nanoparticle Ink[END_REF][START_REF] Paeng | Low-Cost Facile Fabrication of Flexible Transparent Copper Electrodes by Nanosecond Laser Ablation[END_REF]. Secondly, in the process of fabrication of metallic honeycomb electrode through pulsed laser ablation metal thin films, laser fluence at the peripheral of Gaussian spot is between the melting threshold and the ablation threshold of the material. In this case, the temperature gradient drives the molten materials to the outer edge of the spot, and the cylindrical ring or droplet finger structures are formed in the peripheral dewetting zone due to the solidification of transported matter following the laser pulse exposure. (the height is more than twice times thicker than that of the original film) [START_REF] Hong | Nonvacuum, Maskless Fabrication of a Flexible Metal Grid Transparent Conductor by Low-Temperature Selective Laser Sintering of Nanoparticle Ink[END_REF][START_REF] Paeng | Laser wavelength effect on laser-induced photo-thermal sintering of silver nanoparticles[END_REF][START_REF] Qi | Time-resolved analysis of thickness-dependent dewetting and ablation of silver films upon nanosecond laser irradiation[END_REF]. And for oxygen sensitive metal films, such as Cu, Ni and other materials, Gaussian pulse direct ablation process not only causes uneven metallic honeycomb, but the re-oxidized metallic materials can reduce the conductive properties of metallic honeycomb electrodes [START_REF] Paeng | Characteristic time scales of coalescence of silver nanocomposite and nanoparticle films induced by continuous wave laser irradiation[END_REF][START_REF] Paeng | Laser-Induced Reductive Sintering of Nickel Oxide Nanoparticles under Ambient Conditions[END_REF][START_REF] Paeng | Laser wavelength effect on laser-induced photo-thermal sintering of silver nanoparticles[END_REF][START_REF] Qi | Time-resolved analysis of thickness-dependent dewetting and ablation of silver films upon nanosecond laser irradiation[END_REF]. In summary, the roughness of the metallic electrode reduces the light transmittance, and the cylindrical ring, droplet finger structures and the inhomogeneous of the compound metallic electrode increase the leakage of the electronic device, which largely restricts the development of laser direct ablation process for the fabrication of high quality flexible transparent electrode.

In this work, Ag honeycomb FTCEs were produced by a facile cold machining after one-step top-flat square nanosecond LDW ablation of Ag film under ambient conditions. Here, the term "cold machining" refers to the phenomenon wherein ablation occurs just at the square spot without outward radial mass transfer of molten metal, resulting in no structural deformation and damage on the edge of metallic honeycombs. Besides, square metallic honeycombs can effectively improve the areal density according to the circle metallic honeycombs, and the higher areal density results in the higher transparency. In this condition, compare with the Gaussian beam LDW ablation process, the top-flat square LDW ablation process not only maintain Ag mesh with higher electrical conductivity but also higher transparency. The electrical performance of Ag metallic honeycombs FTCEs on polyethylene naphthalate (PEN) substrate is maintained even after bending, folding, twisting, squeezing and ultrasonic bath tests. In addition, the transmittance and conductivity of the Ag metallic honeycombs electrode enable the fabrication of a transparent touch screen panel.

The LDW ablation process was applied on the as-prepared thin silver film using a nanosecond laser (Nd:YVO4, wavelength:532 nm, duration time: 22.5 ns). A schematic of the laser setup for the fabrication of Ag mesh FTCEs is shown in Fig. 1(a). Upon irradiation of nanosecond laser pulses, a top-flat square beam can be modulated by a diffractive optical element, resulting in the fabrication of square honeycomb arrays in silver film on a polyethylene naphthalate (PEN) substrate. In this case, the Ag mesh not only has the superiority of the higher transparency but also maintains the good electronic conductivity of silver. Fig. 1(b) shows a 5 cm × 5 cm flexible transparent conductive electrode on PEN composed of mesh-type Ag with a square honeycomb density of 79% in the red box (2 cm × 2 cm area); no laser ablation of the Ag film on PEN is observed in the blue box. It is noted that this demonstration uses just a 1 kHz laser, and the total laser processing time required for scanning a 2 cm × 2 cm area with a scanning speed of 25 mm/s is only approximately 10 min, which can be possibly shortened by further optimization, such as higher laser repetition and higher scanning speed. A 5 cm × 5 cm Ag mesh electrode could be fabricated within 2-5 s using an array of 30 μm square honeycomb, a laser at 1 MHz pulse repetition, and 10 m/s sample translation speed to write the square honeycomb patterns. In addition, Fig. 1(c) shows the SEM of the patterned Ag FTCE by taking advantage of high-resolution top-flat square LDW ablation processing. Interestingly, due to the uniform of the laser intensity in the top-flat square spot, the measured size of each honeycomb is approximately 30 μm, which is similar with the size of the beam spot, and no structural deformation and damage on the edge of honeycombs that are detrimental to the optical characteristics are observed. The uniformity of Ag mesh upon the top-flat square LDW ablation processing is confirmed by the elemental mapping images of Ag and C, as shown in the insets of Fig. 1(c). It is worth noting that the C elements detected in the background are partly from the flexible PEN substrate. The two images show a clear contrast between Ag and C elements in the electrodes, suggesting that the top-flat square LDW laser irradiation can cleanly removes the irradiated parts by cold machining technology. Because of heat diffusion on the Ag film induced by the laser irradiation, and the Ag ratio at the rim is higher than that at the edge of the honeycomb, which may lead to a reduced conductivity of the electrodes. Some references also mentioned that the smooth and flat metal electrode could improve characteristics of the transparent electrode, such as transparency, conductivity and stability. [1, 2, 24] Furthermore, the atomic force microscopy (AFM) image shows that the height and morphology between the film and the ablated spot after Gaussian and top-flat square beam irradiation, as shown in Fig. 2(c). For the Gaussian beam LDW ablation processing, the nominal thickness of the electrode measured by AFM is approximately 25 nm. Condensed and re-solidified Ag clusters are found near the peripheries of the honeycombs due to the stronger melt expulsion and ablation. In addition, the rim of the ablated honeycomb is around two times thicker than the original film, suggesting outward radial mass transfer of molten metal, as shown in Fig. 2(a). For the top-flat square LDW ablation processing, the rim droplet fingers will not appear in the edge of the spot, and the inner honeycomb is smooth and neat, as shown in Fig. 2(c). It is worth noting that the edges of the top-flat square LDW ablation electrode mesh are sharp, suggesting that this top-flat square LDW ablation process can be applied to a high-resolution electrode fabrication, as shown in Fig. 2(a). In this case, upon the top-flat square ns LDW ablation, square silver honeycomb arrays can be produced in a silver film on a flexible polymer substrate, resulting in higher transparency while maintaining the good electrical conductivity of silver. Besides the advantage of the top-flat LDW ablation processing for the fabrication of Ag mesh electrode, square honeycomb arrays can effectively improve the areal densities according to the circle honeycomb arrays (from 52% to 79%). The areal density is herein defined as the ratio of the honeycomb area after ablation to the total area. By changing the scanning speed, honeycomb arrays with different areal densities can be maintained. For the square arrays, the areal density can be fabricated as high as 79%. However, the areal density of circle arrays is only 52%. The higher areal density result in the higher transparency, in this condition, compare with the Gaussian beam LDW processing, the top-flat square LDW ablation processing can not only maintain Ag mesh with higher electrical conductivity but also higher transparency, which can be defined as cold machining technology. The transmittance of the Ag FTCEs can be controlled by synchronizing the speed of the stage. As-prepared Ag film (square honeycomb areal density=0) yields transmittance of 46% at the wavelength of 550 nm, shown in Fig. 3(a), while the maximum transmittance of 85.1% was achieved when the square honeycomb areal density is 0.79. Fig. 3(b) displays a plot of transmittance at 550 nm versus sheet resistance. The balance between transmittance and sheet resistance can be adjusted by altering the areal density and thickness of the mesh patterns. Due to the smaller line-width and higher square honeycomb density, the 160 nm Ag FTCEs demonstrate high optical transmittance (70.8% at 550 nm wavelength) with very low sheet resistance (5 Ω sq -1 ), as shown in Fig. 3(b). These successful fabrications confirm that the square honeycomb arrays and these key fabrication steps can be reliably scaled down to a sub-micrometer range. Decreasing the thickness of the Ag FTCEs while maintaining same line-width can also effectively improve the transmittance (up to 85.1%). Fig. 3(a) displays the spectra and sheet resistance of a highly transparent Ag FTCEs on a 4 × 4 cm large PEN substrate with different square honeycomb areal density and Ag-mesh thickness of 20, 50 and 160 nm, respectively. Moreover, the suggested top-flat square LDW ablation process method in this work can fabricate electrodes invisible to naked eyes. Because of large line spacing and thin thickness, the Ag FTCE (20 nm) exhibits high optical transmittance (85.1%) and retains low sheet resistance (22.5 Ω sq -1 ). An extremely low sheet resistance of 5 Ω sq -1 was observed for the 160 nm thick Ag FTCEs, and the transmittance at the 550 nm wavelength was still above 70%. In similar fashion, various combinations of optical transparency and sheet resistance can be obtained for various applications by optimizing the geometric parameters of the Ag mesh. To gain further insight into how Ag film thickness affects the overall performance of the Ag FTCEs, a commonly used figure of merit (FoM), namely the ratio of electrical conductance to optical conductance, was calculated for all the FTCEs displayed in Fig. 3(b) by using the following widely accepted expression [START_REF] De | Size Effects and the Problem with Percolation in Nanostructured Transparent Conductors[END_REF][START_REF] Wu | Electrospun Metal Nanofiber Webs as High-Performance Transparent Electrode[END_REF]

Where σOP and σDC are optical conductivity and electrical conductivity of thin metal electrode, respectively. The presented data indicate that the metal thickness has a considerable effect on the sheet resistance and hence on the value of the FoM (σDC/σOP) by increasing the electrical conductivity of a thicker metal mesh without substantially sacrificing transmittance.

As shown in Fig. 4(a), the transparent Ag mesh on PEN was used as a conductor in a circuit to connect a light-emitting diode (LED) under the voltage of 3 V. There was no noticeable brightness change of the LED under folding, twisting and squeezing of the electrode. To identify the long-term mechanical reliability of the Ag mesh electrode, a cyclic bending test was performed. Fig. 4(b) shows the Ag mesh on a PEN substrate test of mechanical stability under cyclic tensile and compressive bending stress. A 2 cm × 5 cm Ag mesh pad was fabricated on the PEN substrate. Bending of the pad is created by moving one end at 60 mm/s of motion speed and a frequency of 1 Hz, yielding a radius of curvature of r = 7 mm at the maximum bending deformation. The resistance of the electrode was measured at the flat state after several bending cycles. After over 10000 cycles, for all three different silver films (20 nm, 50 nm, 160 nm), the measured resistance variation (ΔR/R0) is less than 3%. Moreover, From the SEM images in Fig. 4(b), for all three different thicknesses Ag film, the Ag mesh did not detach from the substrate, and morphology of the Ag mesh did not change much after 10000 cycles bending test.. Since laser irradiation imparts a highly localized temperature field, it is a suitable tool for processing heat-sensitive flexible polymer substrates, and morphological structures of the Ag mesh remained unchanged. This superior bendability is attributed to the strong adhesion of the Ag film (sputtering technology) to the PEN substrate and also to the smooth of Ag square honeycomb arrays. The ultrasonic bath tests performed with Ag mesh electrode further confirmed strong adhesion of the electrodes on the PEN substrate even though the line-width is only 1 μm. As a device demonstration of the Ag mesh electrode, a flexible transparent four-wire resistive touch screen panel was fabricated, as shown in Fig. 5. The 5 cm × 7 cm Ag mesh-type with square density of 79% on the quartz substrate (Rs: 23.2 Ω sq -1 , T: 85.1% at 550 nm) were fabricated as shown in Fig. 5(a). No protective coating layer such as PEDOT:PSS was required for the transparent Ag mesh electrode due to strong adhesion on the PEN or quartz substrate. An ITO-PET film (Rs: 100 Ω sq -1 , T: 86% at 550 nm, Teonex) was used as a counter electrode. Two slips of copper tape were attached to the top and bottom sides of the Ag mesh while another two slips were attached to the left and right sides of the ITO-PET film. Due to the transparency of the Ag mesh electrodes, the colored letters beneath the Touch screen panel (TSP) are visible, as shown in Fig. 5(b). The functionality of the touch screen panel is confirmed by writing the University name was evaluated as displayed in Fig. 5(c).

In summary, flexible-compatible maskless Ag honeycomb electrode was prepared by selective LDW ablation of silver thin films deposited via magnetron sputtering system. Thin (20 nm, 50 nm and 160 nm) Ag honeycombs electrodes with fine morphology were produced due to the cold machining of the top-flat square ns LDW ablation process in the thin film without heat diffusion. Furthermore, compared to Gaussian beam LDW ablation process, the top-flat square ns LDW ablation efficiently reduces the roughness of the metallic electrode and highly increases the light transmittance to 85.1%. In this condition, the top-flat square LDW ablation processing not only maintains Ag mesh with higher electrical conductivity but also higher transparency. Cyclic bending, folding, twisting, squeezing and ultrasonic bath tests confirm robust adhesion of the Ag honeycomb electrodes on both PEN and quartz substrates. In addition, the transmittance and conductivity of the Ag square honeycombs electrode are high enough to fabricate the transparent touch screen panels without any protective layer over the electrodes.

Fig. 1 .

 1 Fig. 1. (a) Fabrication schematics of Ag mesh FTCEs. (b) Photograph of a transparent conductor on a PEN substrate (PEN substrate in the black-boxed region, Ag film in the blue-boxed region and high-density Ag mesh in the red-boxed region), and the background is the Sather Tower of UC Berkeley. (c) Top-view SEM image of Ag mesh-type electrodes on the PEN substrate. The insets show elemental mapping images for Ag and C of the electrodes. (d) Photo-image of several 2 cm  2 cm mesh patterns on the 4-inch quartz substrate, and the number indicates the corresponding honeycomb density of the square mesh patterns.Fig.1(b) shows a 5 cm × 5 cm flexible transparent conductive electrode on PEN composed of mesh-type Ag with a square honeycomb density of 79% in the red box (2 cm × 2 cm area); no laser ablation of the Ag film on PEN is observed in the blue box. It is noted that this demonstration uses just a 1 kHz laser, and the total laser processing time required for scanning a 2 cm × 2 cm area with a scanning speed of 25 mm/s is only approximately 10 min, which can be possibly shortened by further optimization, such as higher laser repetition and higher scanning speed. A 5 cm × 5 cm Ag mesh electrode could be fabricated within 2-5 s using an array of 30 μm square honeycomb, a laser at 1 MHz pulse repetition, and 10 m/s sample translation speed to write the square honeycomb patterns. In addition, Fig.1(c) shows the SEM of the patterned Ag FTCE by taking advantage of high-resolution top-flat square LDW ablation processing. Interestingly, due to the uniform of the laser intensity in the top-flat square spot, the measured size of each honeycomb is approximately 30 μm, which is similar with the size of the beam spot, and no structural deformation and damage on the edge of honeycombs that are detrimental to the optical characteristics are observed. The uniformity of Ag mesh upon the top-flat square LDW ablation processing is confirmed by the elemental mapping images of Ag and C, as shown in the insets of Fig.1(c). It is worth noting that the C elements detected in the background are partly from the flexible PEN substrate. The two images show a clear contrast between Ag and C elements in the electrodes, suggesting that the top-flat square LDW laser irradiation can cleanly removes the irradiated parts by cold machining technology. Because of heat diffusion on the Ag film induced by the laser irradiation, and the Ag ratio at the rim is higher than that at the edge of the honeycomb, which may lead to a reduced conductivity of the electrodes. Some references also mentioned that the smooth and flat metal electrode could improve characteristics of the transparent electrode, such as transparency, conductivity and stability.[1, 2, 24] Fig. 1(d) shows the effect of the square honeycomb arrays on substratebased transmittance of the Ag mesh electrodes on a 4-inch quartz substrate. By optimizing the density and size of the square honeycomb

  Fig. 1. (a) Fabrication schematics of Ag mesh FTCEs. (b) Photograph of a transparent conductor on a PEN substrate (PEN substrate in the black-boxed region, Ag film in the blue-boxed region and high-density Ag mesh in the red-boxed region), and the background is the Sather Tower of UC Berkeley. (c) Top-view SEM image of Ag mesh-type electrodes on the PEN substrate. The insets show elemental mapping images for Ag and C of the electrodes. (d) Photo-image of several 2 cm  2 cm mesh patterns on the 4-inch quartz substrate, and the number indicates the corresponding honeycomb density of the square mesh patterns.Fig.1(b) shows a 5 cm × 5 cm flexible transparent conductive electrode on PEN composed of mesh-type Ag with a square honeycomb density of 79% in the red box (2 cm × 2 cm area); no laser ablation of the Ag film on PEN is observed in the blue box. It is noted that this demonstration uses just a 1 kHz laser, and the total laser processing time required for scanning a 2 cm × 2 cm area with a scanning speed of 25 mm/s is only approximately 10 min, which can be possibly shortened by further optimization, such as higher laser repetition and higher scanning speed. A 5 cm × 5 cm Ag mesh electrode could be fabricated within 2-5 s using an array of 30 μm square honeycomb, a laser at 1 MHz pulse repetition, and 10 m/s sample translation speed to write the square honeycomb patterns. In addition, Fig.1(c) shows the SEM of the patterned Ag FTCE by taking advantage of high-resolution top-flat square LDW ablation processing. Interestingly, due to the uniform of the laser intensity in the top-flat square spot, the measured size of each honeycomb is approximately 30 μm, which is similar with the size of the beam spot, and no structural deformation and damage on the edge of honeycombs that are detrimental to the optical characteristics are observed. The uniformity of Ag mesh upon the top-flat square LDW ablation processing is confirmed by the elemental mapping images of Ag and C, as shown in the insets of Fig.1(c). It is worth noting that the C elements detected in the background are partly from the flexible PEN substrate. The two images show a clear contrast between Ag and C elements in the electrodes, suggesting that the top-flat square LDW laser irradiation can cleanly removes the irradiated parts by cold machining technology. Because of heat diffusion on the Ag film induced by the laser irradiation, and the Ag ratio at the rim is higher than that at the edge of the honeycomb, which may lead to a reduced conductivity of the electrodes. Some references also mentioned that the smooth and flat metal electrode could improve characteristics of the transparent electrode, such as transparency, conductivity and stability.[1, 2, 24] Fig. 1(d) shows the effect of the square honeycomb arrays on substratebased transmittance of the Ag mesh electrodes on a 4-inch quartz substrate. By optimizing the density and size of the square honeycomb

Fig. 2 .

 2 Fig. 2. (a) Schematics of morphology transformations of Ag film upon Gaussian LDW ablation processing and ultrathin Ag film upon top-flat square LDW ablation processing. IGaussian and ISquare are the laser intensity profiles for the Gaussian laser beam and the top-flat square laser beam, respectively. The intensity profile is presented with a dashed line. The solid arrows indicate the transport directions of molten silver upon the laser irradiation. (b) The typical SEM images of the corresponding 20 nm silver film on PEN substrate after Gaussian beam and Top-flat square LDW ablation processes, and fluences of the irradiation laser 1.44 J/cm 2 for Gaussian beam, and 490 mJ/cm 2 for top-flat square beam, and the scale bars are 5 m. (c) AFM image of the Ag FTCEs.Furthermore, the atomic force microscopy (AFM) image shows that the height and morphology between the film and the ablated spot after Gaussian and top-flat square beam irradiation, as shown in Fig.2(c). For the Gaussian beam LDW ablation processing, the nominal thickness of the electrode measured by AFM is approximately 25 nm. Condensed and re-solidified Ag clusters are found near the peripheries of the honeycombs due to the stronger melt expulsion and ablation. In addition, the rim of the ablated honeycomb is around two times thicker than the original film, suggesting outward radial mass transfer of molten metal, as shown in Fig.2(a). For the top-flat square LDW ablation processing, the rim droplet fingers will not appear in the edge of the spot, and the inner honeycomb is smooth and neat, as shown in Fig.2(c). It is worth noting that the edges of the top-flat square LDW ablation electrode mesh are sharp, suggesting that this top-flat square LDW ablation process can be applied to a high-resolution electrode fabrication, as shown in Fig.2(a). In this case, upon the top-flat square ns LDW ablation, square silver honeycomb arrays can be produced in a silver film on a flexible polymer substrate, resulting in higher transparency while maintaining the good electrical conductivity of silver. Besides the advantage of the top-flat LDW ablation processing for the fabrication of Ag mesh electrode, square honeycomb arrays can effectively improve the areal densities according to the circle honeycomb arrays (from 52% to 79%). The areal density is herein defined as the ratio of the honeycomb area after ablation to the total area. By changing the scanning speed, honeycomb arrays with different areal densities can be maintained. For the square arrays, the areal density can be fabricated as high as 79%. However, the areal density of circle arrays is only 52%. The higher areal density result in the higher transparency, in this condition, compare with the Gaussian beam LDW processing, the top-flat square LDW ablation processing can not only maintain Ag mesh with higher electrical conductivity but

Fig. 3 .

 3 Fig. 3. (a) The transmittance spectra of Ag FTCEs with different areal densities (20 nm thicknesses). (b) Plot of sheet resistance versus transmittance at 550 nm wavelength of Ag FTCEs at different square honeycomb areal densities and different thickness of Ag film.The transmittance of the Ag FTCEs can be controlled by synchronizing the speed of the stage. As-prepared Ag film (square honeycomb areal density=0) yields transmittance of 46% at the wavelength of 550 nm, shown in Fig.3(a), while the maximum transmittance of 85.1% was achieved when the square honeycomb areal density is 0.79. Fig.3(b) displays a plot of transmittance at 550 nm versus sheet resistance. The balance between transmittance and sheet resistance can be adjusted by altering the areal density and thickness of the mesh patterns. Due to the smaller line-width and higher square honeycomb density, the 160 nm Ag FTCEs demonstrate high optical transmittance (70.8% at 550 nm wavelength) with very low sheet resistance (5 Ω sq -1 ), as shown in Fig.3(b). These successful fabrications confirm that the square honeycomb arrays and these key fabrication steps can be reliably scaled down to a sub-micrometer range. Decreasing the thickness of the Ag FTCEs while maintaining same line-width can also effectively improve the transmittance (up to 85.1%). Fig.3(a) displays the spectra and sheet resistance of a highly transparent Ag FTCEs on a 4 × 4 cm large PEN substrate with different square honeycomb areal density and Ag-mesh thickness of 20, 50 and 160 nm, respectively. Moreover, the suggested top-flat square LDW ablation process method in this work can fabricate electrodes invisible to naked eyes. Because of large line spacing and thin thickness, the Ag FTCE (20 nm) exhibits high optical transmittance (85.1%) and retains low sheet resistance (22.5 Ω sq-1 ). An extremely low sheet resistance of 5 Ω sq -1 was observed for the 160 nm thick Ag FTCEs, and the transmittance at the 550 nm wavelength was still above 70%. In similar fashion, various combinations of optical transparency and sheet resistance can be obtained for various applications by optimizing the geometric parameters of the Ag mesh. To gain further insight into how Ag film thickness affects the overall performance of the Ag FTCEs, a commonly used figure of merit (FoM), namely the ratio of electrical conductance to optical conductance, was calculated for all the FTCEs displayed in Fig.3(b) by using the following widely accepted expression[START_REF] De | Size Effects and the Problem with Percolation in Nanostructured Transparent Conductors[END_REF][START_REF] Wu | Electrospun Metal Nanofiber Webs as High-Performance Transparent Electrode[END_REF]

Fig. 4 .

 4 Fig. 4. (a) Mechanical durability test with folding, twisting and squeezing with bare hands. Photographs of Ag mesh based FTCE, and lighting of an LED with an external power supply (3V). (b) The cyclic bending test up to 10000 cycles to show electrical and mechanical stability, and the corresponding SEM images showing morphology in the Ag mesh on PEN substrate after repeated tensile bending 10000 cycles) to radius of 7 mm, the honeycomb areal densities for three thickness Ag mesh are all around 70%.

Fig. 5 .

 5 Fig. 5. (a) Schematic diagram of the touch screen panel using Ag mesh on a quartz substrate. (b) Photographs of the four-wire resistive touch screen panel at flat. (c) A touch panel demonstration with the Ag honeycomb on quartz film. Touch screen panel (TSP) attached on a LCD monitor and the schematics of a TSP. Demonstration of the TSP operation fabricated by Ag mesh and ITO-PEN film. "NBU" was written on the TSP.As a device demonstration of the Ag mesh electrode, a flexible transparent four-wire resistive touch screen panel was fabricated, as shown in Fig.5. The 5 cm × 7 cm Ag mesh-type with square density of 79% on the quartz substrate (Rs: 23.2 Ω sq -1 , T: 85.1% at 550 nm) were fabricated as shown in Fig.5(a). No protective coating layer such as PEDOT:PSS was required for the transparent Ag mesh electrode due to strong adhesion on the PEN or quartz substrate. An ITO-PET film (Rs: 100 Ω sq -1 , T: 86% at 550 nm, Teonex) was used as a counter electrode. Two slips of copper tape were attached to the top and bottom sides of the Ag mesh while another two slips were attached to the left and right sides of the ITO-PET film. Due to the transparency of the Ag mesh electrodes, the colored letters beneath the Touch screen panel (TSP) are visible, as shown in Fig.5(b). The functionality of the touch screen panel is confirmed by writing the University name was evaluated as displayed in Fig.5(c).

Funding.

  Acknowledge the National Natural Science Foundation of China (61705117); Natural Science Foundation of Ningbo (2018A610043); and Chinese Scholars Council. And acknowledge the support of 3315 innovation team, Ningbo city. It was also support by K.C. Wong Magna Fund in Ningbo University.


Arnaud Autret

Sandra Martin-Latil

Cynthia Brisac

Laurence Mousson

Florence Colbère-Garapin

Bruno Blondel
email: bblondel@pasteur.fr

Early phosphatidylinositol 3-kinase/Akt pathway activation limits poliovirus-induced JNK-mediated cell death

PV-induced apoptosis seems to play a major role in tissue injury in the central nervous system (CNS). We have previously shown that this process involves PV-induced Bax-dependent mitochondrial dysfunction mediated by early JNK activation in IMR5 neuroblastoma cells.

We show here that PV simultaneously activates the phosphatidylinositol 3-kinase (PI3K)/Akt survival signaling pathway in these cells, limiting the extent of JNK activation, and thereby cell death. JNK inhibition is associated with PI3K-dependent negative regulation of the apoptosis signal-regulating kinase 1 (ASK1), which acts upstream from JNK in PV-infected IMR5 cells. In poliomyelitis, this survival pathway may limit the spread of PV-induced damage in the CNS.

Poliovirus (PV), from the Picornaviridae family, causes paralytic poliomyelitis -a disease in which the motor neurons are destroyed in association with PV replication. PV consists of a single-stranded positive RNA genome surrounded by a nonenveloped icosahedral protein capsid. The human PV receptor, CD155, and its simian counterparts belong to the immunoglobulin superfamily [START_REF] Koike | The poliovirus receptor protein is produced both as membrane-bound and secreted forms[END_REF][START_REF] Koike | A 2nd gene for the African green monkey poliovirus receptor that has no putative Nglycosylation site in the functional N-terminal immunoglobulin-like domain[END_REF][START_REF] Mendelsohn | Cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily[END_REF] and are related to the nectin family of adhesion molecules [START_REF] Lopez | Complementary DNA characterization and chromosomal localization of a human gene related to the poliovirus receptor-encoding gene[END_REF][START_REF] Takahashi | Nectin/PRR: an immunoglobulin-like cell adhesion molecule recruited to cadherin-based adherens junctions through interaction with Afadin, a PDZ domain-containing protein[END_REF].

PV is mostly transmitted via the fecal-oral route. It first infects the oropharynx and the digestive tract, and then spreads to the central nervous system (CNS) in which it mostly targets motor neurons. Studies in mouse models have shown that PV-infected motor neurons in the spinal cord die by apoptosis [START_REF] Blondel | Poliovirus, pathogenesis of poliomyelitis, and apoptosis[END_REF][START_REF] Girard | Poliovirus induces apoptosis in the mouse central nervous system[END_REF]. PV-induced apoptosis therefore seems to play a major role in the tissue injury occurring in the CNS.

PV triggers apoptosis in vitro in tissue cultures of human colon carcinoma cells (CaCo-2) (4), promonocytic cells (U937) [START_REF] Lopez-Guerrero | Poliovirus induces apoptosis in the human U937 promonocytic cell line[END_REF], dendritic cells [START_REF] Wahid | Dendritic cells and macrophages are productively infected by poliovirus[END_REF], murine L cells expressing CD155 [START_REF] Gosselin | Poliovirusinduced apoptosis is reduced in cells expressing a mutant CD155 selected during persistent poliovirus infection in neuroblastoma cells[END_REF][START_REF] Romanova | Variability in apoptotic response to poliovirus infection[END_REF], HeLa cells [START_REF] Belov | The major apoptotic pathway activated and suppressed by poliovirus[END_REF][START_REF] Tolskaya | Apoptosis-inducing and apoptosispreventing functions of poliovirus[END_REF] and cultures of mixed mouse primary nerve cells [START_REF] Couderc | An ex vivo murine model to study poliovirus-induced apoptosis in nerve cells[END_REF] from the cerebral cortex of mice transgenic for CD155. Analyses of the apoptotic pathways induced following PV infection in several cell lines have demonstrated that mitochondria are key actors of PV-induced apoptosis. In particular, mitochondrial outer membrane permeabilization (MOMP) following PV infection leads to a loss of mitochondrial transmembrane potential and the release of proapoptotic molecules, including cytochrome c, from the mitochondria to the cytosol [START_REF] Belov | The major apoptotic pathway activated and suppressed by poliovirus[END_REF][START_REF] Gosselin | Poliovirusinduced apoptosis is reduced in cells expressing a mutant CD155 selected during persistent poliovirus infection in neuroblastoma cells[END_REF]. We recently demonstrated that MOMP in PVinfected neuronal IMR5 cells was dependent on Bax, a proapoptotic member of the Bcl-2 family. Bax activation was mediated by c-Jun NH2-terminal kinase (JNK) phosphorylation after PV infection [START_REF] Autret | Poliovirus induces Baxdependent cell death mediated by c-Jun NH2-terminal kinase[END_REF]. JNK activation occurred early after PV infection whereas apoptotic features were observed later in PV-infected cells. These events may involve a balance between pro-and antiapoptotic signals following PV infection. Pro-and antiapoptotic events potentially acting in synergy or competing with each other during the reproduction cycle of PV have been described by Agol's group [START_REF] Agol | Competing death programs in poliovirusinfected cells: commitment switch in the middle of the infectious cycle[END_REF][START_REF] Tolskaya | Apoptosis-inducing and apoptosispreventing functions of poliovirus[END_REF]. However, the mechanisms involved in maintaining this delicate balance remain unclear.

Cells become committed to undergoing apoptosis in response to a collection of multiple survival and death signals. The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays a crucial role in the transmission of survival signals in various cell types [START_REF] Datta | Cellular survival: a play in three Akts[END_REF][START_REF] Lawlor | PKB/Akt: a key mediator of cell proliferation, survival and insulin responses?[END_REF], including neurons [START_REF] Dudek | Regulation of neuronal survival by the serine-threonine protein kinase Akt[END_REF]. PI3K activates its downstream effector, the serine-threonine kinase Akt (also known as protein kinase B, PKB) by promoting its phosphorylation at the residues Thr308 and Ser473. Activated Akt then phosphorylates various substrates, activating antiapoptotic factors and inactivating proapoptotic factors. The role of PI3K/Akt in the regulation of cell survival and apoptosis in a number of viral infection models [START_REF] Cooray | The pivotal role of phosphatidylinositol 3-kinase-Akt signal transduction in virus survival[END_REF][START_REF] Dahl | Evidence of a role for phosphatidylinositol 3-kinase activation in the blocking of apoptosis by polyomavirus middle T antigen[END_REF][START_REF] Ehrhardt | Bivalent role of the phosphatidylinositol-3-kinase (PI3K) during influenza virus infection and host cell defence[END_REF][START_REF] Lee | Flavivirus activates phosphatidylinositol 3-kinase signaling to block caspase-dependent apoptotic cell death at the early stage of virus infection[END_REF][START_REF] Meili | Protein kinase B/Akt is activated by polyomavirus middle-T antigen via a phosphatidylinositol 3kinase-dependent mechanism[END_REF], including infection with coxsackievirus B3 [START_REF] Esfandiarei | Protein kinase B/Akt regulates coxsackievirus B3 replication through a mechanism which is not caspase dependent[END_REF], rhinovirus [START_REF] Newcomb | Phosphatidylinositol 3-kinase is required for rhinovirusinduced airway epithelial cell interleukin-8 expression[END_REF], foot-and-mouth disease virus [START_REF] Peng | VP1 of foot-and-mouth disease virus induces apoptosis via the Akt signaling pathway[END_REF] and enterovirus 71 [START_REF] Tung | EV71 induces VCAM-1 expression via PDGF receptor, PI3-K/Akt, p38 MAPK, JNK and NF-kappaB in vascular smooth muscle cells[END_REF][START_REF] Wong | Phosphorylation of PI3K/Akt and MAPK/ERK in an early entry step of enterovirus 71[END_REF] -all members of the Picornaviridae family -has recently been investigated.

PV activates the PI3K/Akt survival signaling pathway in IMR5 cells

We began by determining whether PV infection of IMR5 neuroblastoma cells resulted in Akt activation. IMR5 cells were infected with PV as previously described [START_REF] Autret | Poliovirus induces Baxdependent cell death mediated by c-Jun NH2-terminal kinase[END_REF]. Briefly, the growth medium (DMEM supplemented with 10% FBS) was discarded. The virus was then added to monolayers at a multiplicity of infection (MOI) of ten 50% tissue culture infective dose units (TCID 50 ) per cell (this MOI was used for all assays performed in this study).

Adsorption was allowed to proceed for 30 min at 37°C in humidified air containing 5% CO 2 .

Cells were then washed twice with serum-free medium to remove unbound particles and incubated with fresh DMEM supplemented with 10% FBS at 37°C. The virus was allowed to grow for the indicated times. Time zero postinfection (p.i) corresponds to the inoculation time point. Mock-infected cells were used as a negative control. As previously described (6), both adherent and detached cells were taken into account in all experiments. Kinetics of Akt phosphorylation at serine 473 (Ser473), which is required for full Akt activation (3), was investigated in mock-and PV-infected cells. Whole-cell lysates were analyzed at the indicated times p.i. by Western blotting with a specific anti-phospho (Ser473)-Akt antibody (Fig. 1A).

We checked for equal protein loading on the total Akt Western blot. The amount of phosphorylated Akt increased until 30 min p.i., and then decreased; at 4 h p.i., the amount of phosphorylated Akt present was similar to that in mock-infected cells analyzed at the same time point. To check that the virus stock used in this study did not contain host-derived components that may activate Akt signaling pathway, we depleted the virus suspension of PV using an anti-PV antibody and infected cells with either the depleted or non-depleted suspension. In contrast to cells infected with the non-depleted stock, no Akt activation (30 min p.i.) was detected in cells treated with the depleted suspension (Fig. 1A, bottom, left). We also checked that poliovirus, purified by isopycnic CsCl gradient centrifugation [START_REF] Blondel | Detection by monoclonal antibodies of an antigenic determinant critical for poliovirus neutralization present on VP1 and on heat-inactivated virions[END_REF], could promote Akt activation (30 min p.i.), at an efficiency similar to that obtained with the virus preparations used in this study (Fig. 1A, bottom, right). We then investigated whether Akt activation in response to PV infection occurred through the PI3K pathway, by treating IMR5 cells with a specific PI3K inhibitor, wortmannin [START_REF] Arcaro | Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses[END_REF], at a concentration of 100 nM and 500 nM, 2 h before mock or virus infection. The concentration of the inhibitor was maintained during the adsorption period and PV infection. Cell lysates were collected 30 min after infection and subjected to Western blot analysis for the detection of Akt phosphorylation (Fig. 1B, top). Wortmannin inhibited Akt phosphorylation at both concentrations without altering total Akt levels. The activation of Akt in response to PV infection was illustrated by immunofluorescence staining, 30 min p.i., with the same anti-phospho (Ser473)-Akt antibody.

Representative staining patterns for mock-infected and PV-infected IMR5 cells treated with wortmannin or left untreated are presented (Fig. 1B, bottom). As expected, immunofluorescence staining was detected only in infected cells in the absence of wortmannin. Thus, the rapid PV-induced phosphorylation of Akt involves a PI3K-dependent mechanism.

We investigated whether PV adsorption onto IMR5 cells induced Akt activation in the absence of PV replication by assessing Akt phosphorylation after the addition of UVinactivated PV (UV cross-linked at 6,000 µJ/cm 2 ) to IMR5 cells at a dilution corresponding to an MOI of 10 TCID 50 per cell [START_REF] Autret | Poliovirus induces Baxdependent cell death mediated by c-Jun NH2-terminal kinase[END_REF]. The complete abolition of viral infectivity by UV light treatment was confirmed by titration assay with undiluted viral suspension. We also checked that UV inactivation did not modify virus adsorption on cells, by comparing the binding efficiency of infectious and UV light-treated PV labeled with [ 35 S]methionine (data not shown). Akt phosphorylation was induced in IMR5 cells 30 minutes after the addition of UVinactivated PV, with an efficiency similar to that observed with infectious PV (Fig. 2). Thus, PV-cell receptor interaction alone is sufficient to induce Akt phosphorylation in the absence of viral replication.

PI3K/Akt signaling pathway limits the amplitude of Bax activation, cytochrome c release and apoptosis in PV-infected IMR5 cells

We assessed the role of the PI3K/Akt signaling pathway in regulating the mitochondrial pathway of apoptosis in PV-infected cells, by blocking PI3K activation with wortmannin. The mitochondrial pathway is regulated by members of the Bcl-2 family, including the proapoptotic protein Bax, which promotes the release of cytochrome c. Baxmediated cell death involves several well-controlled steps, including a conformational change resulting in exposure of the NH2-terminus. Mock-and PV-infected IMR5 cells were left untreated or were treated with 100 nM wortmannin for 2 h before PV infection. The concentration of the inhibitor was maintained throughout both PV adsorption and replication. At 8 h p.i., a time point at which Bax activation is known to occur in PV-infected cells (6), whole-cell lysates were prepared in a lysis buffer containing 1% of the zwitterionic detergent CHAPS, which has no effect on Bax conformation [START_REF] Hsu | Bax in murine thymus is a soluble monomeric protein that displays differential detergent-induced conformations[END_REF]. Bax was then immunoprecipitated with an anti-Bax antibody (6A7) that specifically recognizes Bax protein with an exposed NH2 terminus. The Bax protein immunoprecipitated from mock-and PV-infected cells was visualized by Western blotting (Fig. 3A, top). No activated Bax was detected in the immunoprecipitates from mock-infected cells. Consistent with our previous report [START_REF] Autret | Poliovirus induces Baxdependent cell death mediated by c-Jun NH2-terminal kinase[END_REF], Bax was immunoprecipitated with the 6A7 antibody at 8 h p.i., indicating that PV infection was responsible for inducing the change in Bax conformation. Wortmannin enhanced Bax activation in IMR5-infected cells, without affecting the total amount of Bax (Fig. 3A, bottom). The effect of wortmannin on cytochrome c efflux from the mitochondria of PVinfected cells was also investigated. Whole-cell extracts from mock-or PV-infected cells were fractionated at 8 h p.i., to separate the cytosolic fraction from the heavy membrane fraction, including mitochondria, as previously described [START_REF] Autret | Poliovirus induces Baxdependent cell death mediated by c-Jun NH2-terminal kinase[END_REF]. Cytochrome c release was analyzed by Western blotting the cytosolic fraction. Much more cytochrome c was released in response to PV infection in cells treated with wortmannin than in untreated infected cells (Fig. 3B). These results suggest that PI3K may inhibit Bax-dependent MOMP during the PV infection of IMR5 cells.

We investigated the possible involvement of PV-mediated PI3K activation in the inhibition of apoptosis, by analyzing the kinetics of apoptosis in mock infected and infected cells treated or not treated with the specific PI3K inhibitor, wortmannin (Fig. 3C). Adherent and detached cells were harvested at the indicated times p.i. and apoptosis was analyzed by assessing chromatin condensation and fragmentation by flow cytometry after acridine orange (AO) nuclear dye staining, as previously described [START_REF] Autret | Poliovirus induces Baxdependent cell death mediated by c-Jun NH2-terminal kinase[END_REF]. We found that levels of PV-induced apoptosis were higher in infected cells treated with wortmannin than in untreated infected cells. To confirm the role of PI3K/Akt signaling pathway in limiting PV-induced apoptosis, we down-regulated Akt expression with a specific siRNA. Western blot analysis with a specific antibody showed that Akt expression in IMR5 cells transfected with Akt siRNA was significantly weaker than in cells transfected with a nontargeted control siRNA (Fig. 3D,left).

As expected, following PV infection (8 h p.i.), apoptosis levels were higher in Akt knockdown cells than in nontargeted control siRNA-transfected cells (Fig. 3D,right). These results suggest that PI3K/Akt pathway plays a role in inhibiting the mitochondrial apoptotic pathway in PV-infected IMR5 cells.

The PI3K/Akt signaling pathway does not affect PV growth, but delays PV release

We evaluated the effects of PI3K/Akt signaling on the amount of total virus produced in IMR5 cells, by determining the kinetics of total virus yield by TCID 50 assays in the presence or absence of wortmannin. PI3K/Akt pathway inhibition had no effect on the total amount of virus produced (Fig. 4). As PV-induced apoptosis levels were higher in infected cells treated with wortmannin than in untreated infected cells, we assessed the possible effects of the increase in apoptosis levels on externalization of the virus. Viruses were released earlier in the presence of wortmannin (Fig. 4). Thus, PI3K/Akt seems to delay viral release without affecting virus production.

The PI3K/Akt signaling pathway limits JNK activation in PV-infected cells

We have shown that Bax-dependent activation of the mitochondrial pathway of apoptosis is mediated by early JNK activation [START_REF] Autret | Poliovirus induces Baxdependent cell death mediated by c-Jun NH2-terminal kinase[END_REF]. JNK activation peaks 30 min p.i and then decreases in IMR5 neuroblastoma cells. It is possible that the PI3K/Akt pathway down regulates the JNK pathway, as recently reported in nonviral models [START_REF] Aikin | Cross-talk between phosphatidylinositol 3-kinase/AKT and c-jun NH2-terminal kinase mediates survival of isolated human islets[END_REF][START_REF] Kim | Akt phosphorylates and negatively regulates apoptosis signal-regulating kinase 1[END_REF].

We assessed the effects of PI3K/Akt on JNK activation in PV-infected cells, by treating cells with wortmannin. JNK activation was investigated 30 min p.i., by Western blotting whole-cell lysates with an antibody against phosphorylated forms of JNK (Fig. 5A).

As expected, phosphorylated JNK was detected 30 min p.i.. Larger amounts of phosphorylated JNK were found in infected cells treated with wortmannin than in untreated cells. Thus, activation of the PI3K/Akt pathway limits JNK activation in PV-infected IMR5 cells.

JNK activation is limited by the Akt-mediated phosphorylation of ASK1 in PV-infected cells

We then examined the possibility that a kinase, upstream of JNK, was inhibited by Akt, causing the observed limited JNK phosphorylation in PV-infected cells. Apoptosis signal-regulating kinase 1 (ASK1) has been shown to be a key regulator of the JNK pathway amenable to inhibition by Akt-mediated phosphorylation at Ser83 in nonviral systems [START_REF] Aikin | Cross-talk between phosphatidylinositol 3-kinase/AKT and c-jun NH2-terminal kinase mediates survival of isolated human islets[END_REF][START_REF] Kim | Akt phosphorylates and negatively regulates apoptosis signal-regulating kinase 1[END_REF].

We assessed the possible involvement of ASK1 in JNK activation in PV-infected IMR5 cells, by down-regulating ASK1 expression using specific siRNA [START_REF] Saadatzadeh | Oxidant hypersensitivity of Fanconi anemia type C-deficient cells is dependent on a redox-regulated apoptotic pathway[END_REF]. Western blot analysis with a specific antibody showed that ASK1 levels were significantly lower in IMR5 cells transfected with ASK1 siRNA than in cells transfected with a nontargeted control siRNA (Fig. 5B, left).

Moreover, following PV infection, JNK activation in ASK1 knockdown cells was weaker than in cells transfected with the nontargeted control siRNA (Fig. 5B,right). Thus, ASK1 plays an important role in JNK activation following PV infection in IMR5 cells.

We then investigated the possible limitation of ASK1 activity by PI3K/Akt-mediated phosphorylation at Ser83 in PV-infected cells. The kinetics of ASK1 phosphorylation at Ser83 in PV-infected cells was analyzed by Western blotting with a specific antibody against phosphorylated ASK1 (Fig. 5C). A transient increase in the level of ASK1 phosphorylation was evident 30 minutes after infection, consistent with the pattern of Akt activation. Furthermore, treatment of the cells with the PI3K inhibitor wortmannin abolished the increase in ASK1 phosphorylation in PV-infected cells (Fig. 5D). Altogether, these results indicate that the PI3K/Akt pathway negatively regulates JNK activation by phosphorylating and inactivating ASK1 in PV-infected IMR5 cells.

This study provides evidence that the early PI3K/Akt survival pathway limits the magnitude of PV-induced JNK activation and cell death in IMR5 cells. We previously showed that PV-cell receptor interaction alone is sufficient to induce JNK phosphorylation, as for Akt activation. However, we also showed that JNK phosphorylation is necessary, but not sufficient, to trigger apoptosis that seems to require the active replication of PV. As previously reported by Agol's group [START_REF] Agol | Competing death programs in poliovirusinfected cells: commitment switch in the middle of the infectious cycle[END_REF][START_REF] Tolskaya | Apoptosis-inducing and apoptosispreventing functions of poliovirus[END_REF], several different courses of events may influence apoptosis in PV-infected cells between 30 min and 6-8 h p.i. These events may involve the interplay between cellular and viral proteins [START_REF] Barco | Poliovirus Protease 3Cpro Kills Cells by Apoptosis[END_REF][START_REF] Dodd | Poliovirus 3A protein limits interleukin-6 (IL-6), IL-8, and beta interferon secretion during viral infection[END_REF][START_REF] Goldstaub | Poliovirus 2A Protease Induces Apoptotic Cell Death[END_REF][START_REF] Neznanov | Unstable receptors disappear from cell surface during poliovirus infection[END_REF][START_REF] Neznanov | Poliovirus protein 3A inhibits tumor necrosis factor (TNF)-induced apoptosis by eliminating the TNF receptor from the cell surface[END_REF][START_REF] Weidman | Poliovirus 3C protease-mediated degradation of transcriptional activator p53 requires a cellular activity[END_REF]. Thus, the early PI3K/Akt survival pathway seems to act upstream of this unidentified interplay. The PI3K/Akt pathway has been shown to play an antiapoptotic role in several viral infections [START_REF] Cooray | The pivotal role of phosphatidylinositol 3-kinase-Akt signal transduction in virus survival[END_REF]. However, this is the first report, to our knowledge, of the limitation of JNK activation by PI3K/Akt mediating a survival pathway during a viral infection. We have also shown that the cross-talk between the PI3K/Akt and JNK pathways involved ASK1 inhibition. In poliomyelitis, this survival pathway may limit the spread of PV-induced damage in the CNS.

FIGURE LEGENDS

Fig. 1 .

 1 Fig. 1. PV induces early Akt phosphorylation in a PI3K-dependent manner in IMR5 neuroblastoma cells (A) Kinetics of Akt activation in PV-infected neuronal cells. (Top) Akt activation was analyzed in whole-cell lysates at the indicated times p.i., by Western blotting with a specific anti-phospho (Ser473)-Akt antibody (Cell Signaling). Whole-cell lysates from mock-infected cells were analyzed at 30 min (first lane) and 240 min (last lane) post mock-infection, respectively. Blots were then stripped and reprobed with an antibody recognizing all forms of Akt (Cell Signaling), to confirm equal protein loading. (Bottom) Western blot analyses of Akt activation 30 min p.i.. (Left) Cells were infected with viral stock (PV) or viral stock depleted of PV (PV depleted ) with anti-PV antibody. (Right) Cells were infected with viral stock (PV) or CsCl-purified PV (PV purified ). (B) Inhibition of Akt phosphorylation during PV infection in

Fig. 2 .

 2 Fig. 2. UV-inactivated PV induces early Akt activation in IMR5 cells. Akt activation was analyzed by Western blotting whole-cell lysates from cells infected with infectious or UVinactivated PV (30 min p.i.) with specific anti-phospho (Ser473)-Akt antibody. Blots were then stripped and reprobed with an antibody recognizing all forms of Akt, to confirm equal protein loading.

Fig. 3 .

 3 Fig. 3. Inhibition of the PI3K/Akt signaling pathway enhances PV-induced apoptosis in IMR5 cells (A) Enhancement of Bax activation in PV-infected cells treated with wortmannin. (Top) Cells were uninfected or infected with PV (8 h p.i.) in the presence or absence of wortmannin (100 nM). Cells were lysed in immunoprecipitation buffer. Conformationally active Bax protein was immunoprecipitated (IP) with anti-Bax 6A7 antibody (Santa-Cruz) and precipitates were immunoblotted with anti-Bax antibody. The asterisk indicates immunoglobulin light chains.

Fig. 4 .

 4 Fig. 4. Effect of PI3K/Akt signaling inhibition on PV growth and externalization IMR5 cells were infected with PV in the presence or absence of wortmannin (100 nM). Total virus yield (extracellular and intracellular) was determined by TCID 50 assay at the indicated times after three cycles of freezing and thawing to release intracellular viruses. Extracellular virus titer was determined from the supernatant of PV-infected cells at the indicated times after the removal of detached cells by centrifugation. Each point represents the mean virus titers for two independent experiments. Standard errors of the mean are indicated, *P<0.05 by a Student t test comparing untreated to treated IMR5 cells.

Fig. 5 .

 5 Fig. 5. The PI3K/Akt signaling pathway limits JNK activation by promoting ASK1 phosphorylation in PV-infected IMR5 cells (A) JNK activation levels are higher in PV-infected cells treated with wortmannin. Cells were uninfected or infected with PV (30 min p.i.) in the presence or absence of wortmannin (100 nM). JNK activation was analyzed in whole-cell lysates, by Western blotting with a specific anti-phospho (Thr183/Tyr185)-JNK (p46 [JNK1] and p54 [JNK2/3]) antibody, as previously described (6). Blots were then stripped and reprobed with an antibody recognizing all forms of JNK, to confirm equal protein loading. (B) Inhibition of JNK activation after the knockdown of ASK1 expression in PV-infected IMR5 cells. (Left) IMR5 cells were transfected with ASK1 siRNA (37) or nontargeted control siRNA (Cell Signaling) or left untreated. ASK1 protein was then assayed by immunoblotting with extracts from nontargeted control siRNA-transfected, ASK1 siRNA-transfected or untreated cells. Actin was used as a protein loading control. (Right) Untreated, nontargeted control and ASK1 siRNA transfected

We thank S. Susin and V. Yuste (Institut Pasteur, Paris, France) for providing IMR5 cells and F. Delpeyroux (Institut Pasteur, Paris, France) for the anti-PV antibody. We also thank J.M. Panaud (Institut Pasteur, Paris, France) for assistance with fluorescent microscopy. A. A. was supported by grants from the Ministère de l'Education Nationale, de la Recherche et de la

Technologie. This work was supported by grants from the Institut Pasteur (PTR 120) and Danone Research, Centre Daniel Carasso. IMR5 cells were uninfected or infected with PV. JNK activation was analyzed (30 min p.i.) in whole-cell lysates, by Western blotting with a specific anti-phospho (Thr183/Tyr185)-JNK antibody. Blots were then stripped and reprobed with an antibody recognizing all forms of JNK, to confirm equal protein loading. Phosphorylated JNK protein levels were determined by densitometry and plotted as the ratios, relative to the levels of total JNK. Phosphorylated phosphorylation by the PI3K/Akt pathway inhibitor wortmannin. Cells were uninfected or infected with PV in the presence or absence of wortmannin (100 nM). ASK1 phosphorylation was analyzed (30 min p.i.) in whole-cell lysates by Western blotting with a specific antiphospho (Ser83)-ASK1 antibody. Blots were then stripped and reprobed with an antibody recognizing all forms of ASK1, to confirm equal protein loading. Phosphorylated ASK1 protein levels were determined by densitometry, and plotted as the ratios relative to the levels of total ASK1.


Melissa E Maldonado

Avishek Das

Anderson S L Gomes
email: anderson.lgomes@ufpe.br

Anton A Popov

Sergey M Klimentov

Andrei V Kabashin

Melissa Maldonado Cantillo

Nonlinear photoacoustic response of suspensions of laser-synthesized plasmonic titanium nitride nanoparticles

Titanium nitride (TiN) nanoparticles (NPs) present an alternative plasmonic material to noble metal counterparts, with a red-shifted plasmonic extinction band (600-800 nm) compared to Au and Ag NPs (520-550 nm and 400-420 nm, respectively) [1][2][3]. This feature makes them an attractive candidate for a variety of applications such as solar heat transducers [START_REF] Ishii | Titanium Nitride Nanoparticles as Plasmonic Solar Heat Transducers[END_REF], waveguides in the telecommunication range [5], functional arrays [6], electrochemical supercapacitors [7], etc. Biomedicine looks as one of main beneficiaries of such remarkable plasmonic absorption, falling in the first biological transparency window (630-900 nm) even for relatively small NP sizes. As an example, TiN NPs have been successfully used as photothermal agents for cancer therapy and in vivo imaging [8][9][10]. The mechanism enabling such biomedical applications arises from linear optical absorption near the plasmonic resonance, which provides contrast in bioimaging [11] and initiate a photothermal effect [12]. On the other hand, nonlinear optical (NLO) responses measured by optical Z-scan or other techniques can acquire important information on nonlinear absorption (NLA) and other characteristics, which can also help to unravel novel prospects for the development of imaging and therapy modalities. Such an expectation is confirmed by results of recent optical Z-scan studies reporting a strong NLO response using TiN NPs [13,14].

Photoacoustic (PA) response of absorptive materials provides another attractive opportunity for NPs characterization, which arises from the transformation of absorbed photons into heat. Such a phenomenon forces microscopic expansions and subsequent contractions (after cooling), which in turn generate acoustic waves in the surrounding medium. There is a myriad of applications of photoacoustic techniques, from spectroscopy [15] to multiscale imaging and tomography [16]. When the photoacoustic process takes place in a liquid ambience, nanobubbles and microbubbles can also be generated [17,18]. Photoacoustic Z-scan (PAZ-scan) presents a modification of optical Z-scan, which detects the generation of photoacoustic waves in the NLO regime, while an optical pulse is nonlinearly absorbed by the sample and converted into sound waves [19]. The method has advantages over the optical Z-scan, since it relies solely on the material's absorption, can work with opaque samples and be used with a wide range of excitation wavelength, while scattering does not contribute to the recorded signals. An advanced implementation of this technique, termed OPAZ, combines optical Zscan and PAZ-scan to obtain a better understanding of the NLA in optical materials [20].

In this letter, for the first time we report a quantitative characterization of nonlinear photoacoustic response (NPR) of suspensions of laser-synthesized TiN NPs using OPAZ-scan. The lasersynthesized NPs were chosen as a new appealing object for biomedical applications, combining unique physico-chemical characteristics (spherical shape, controllable size under low size dispersion, absence of toxic impurities, high stability in aqueous or organic solvents, etc.) [21] with high safety in biological models in vitro and in vivo [22], which makes them advantageous compared to counterparts prepared by conventional methods, including chemical synthesis [1], epitaxy growth [6], electrochemical [7] and hydrothermal synthesis [8]. We measured effective nonlinear absorption coefficients βPA, eff and discuss th of e directly implies ess. The photoaco to absorption w h absorption and ortant to verify t which can be ca produced in eq. 1 cient, A is the op s. Using linear ab ntly, the mass erials having a m Au nanorods wer extinction [30]). n Fig. 3. As the (OPO) which 0 nm and 800 nt power was cope, used to two channels. D1) to control to the sample onal optical Zns, in our case e, which was ong the Z-axis. 5 degrees to Z ed water for ted using a 10 U) in front of smitted beam rement of the z=0) was w0 = en Z-scan and . To calculate the bsorptive respon urve using the eq. gap energy was c films [13]. To ind n band of TiN, the gy (Eg). In this wo V) were lower tha and two-photononse the plasmo bution of plasmo velength was in t d results, we can nter of the plasm rly higher than a ct did not appea all scattering effe fficient obtained plasmonic enhan similar to that in ect for the NLA tal results (dots) NP with the O can measuremen e, simultaneously e nonlinear coeff se, we employed (2) [33]: ∑ calculated to be ab duce a transition e photon energy m ork, the excitation an the Eg.

en by [17,18], t:

(5)

effects can be 34], or due to the effect of nts. Since the r of those two scattering, we bbles from the NLA coefficient n in table I. We nfluence of the s detected by Z-scan, which although one re in different oral excitation om table I, the r the TiN NPs se obtained in on wavelength gime [14], the an our values he absence of s not a colloid. om the optical ed from the Z-scan is not conclude that is wavelength grows to βPA, eff he increase of ates significant on, or, in other to increase of s beneficial for her nonlinear nfirms the role of plasmonic enhancement. On the other hand, one important issue is related to the fact that in the photoacoustic measurements, the photons absorbed by TiN NPs are transformed into heat, which is very likely to create microbubbles. In order to clarify the role of such microbubbles, time resolved studies should be performed, as in [16,17], where microbubbles in gold colloids were studied. Therefore, the NLA values obtained in our work could be affected by this phenomenon, and further experiments are required to verify this possibility.

Table I Calculated values of the NLA coefficient for TiN NPs in this work and comparison with literature values

A Excitation intensity one order of magnitude higher than in the present work. B 52nm thick thin film.

In conclusion, effective values of optically induced nonlinear photoacoustics response of suspensions of 40nm TiN NPs were reported for the first time using a combined technique for simultaneous optical and photoacoustic detection. Using the OPAZscan method, it was shown that nonlinear scattering can affect the results, as a loss mechanism influencing the nonlinear absorption. Furthermore, bubble formation due to heating cannot be ruled out and further experiments are required to clarify this issue. Such bubbling effects are important for NPs mediated laser ablation of biological tissues and can be used for potential theranostics applications [16]. The plasmonic enhancement in the TiN NPs can also be exploited for biomedical applications such as photothermal therapy [9,10,21], which can be extended by serial PA tomography [35]

  Fig sca th SE Str sca Te In 1b dis ma ma co an

Fig. 3

 3 Fig. 3. E photoac The r left colu differen simulta right co ranging was mu reprodu scan sig (rather naturall apertur To u energy the well

  Fig NL ph wi

  coustic signal for malized by the faras in our case, t ising from nonlin crobubbles[16- neglected in su t measure the inf e PAZ-scan is no tiate the contribu ation, the values f qs.(1 -5). These A coefficient, βPA hanisms leading also shows resul her literature val comparing the d lm) and differen ve comparison c ed from optical Z e same order of m e duration, but d thin films in the order of magnit d regime. One re and bubble form ect is that the ob so not far from As mentioned g effect. Therefo ay slightly affects βPA, eff ~ 1.36 βOZ-SC 00nm it is βPA, eff ãr the plasmonic r ion of absorption in the plasmon increase of scatte of TiN NPs co onic band centra tional to the NLA is the effectiv L is the sample t n eq. (2), the effec the acoustic sign icient , i.e., d Γ is the Grüneis perties and the ndent of the scatte e light absorptive tic signal is prod cident light and bsorption by NPs e to their high effective nonline r PAZ-scan is give -

  , as demonstrated with gold NP.

	Sam ple	Preparatio n method/ solvent	λ , nm	NP size, nm	βPA, eff ×10 -8 , cm/W	βOZ-SCAN ×10 -8 , cm/W	Ref (τexc)
					3.27
	TiN	Laser ablation/ acetone	600 700 800	40 40 40	±0.17 6.41 ±0.32 3.22	2.40 1.04 1.23	This work (5 ns)
					±0.16
	TiN	Commerci al powder	532	55	---	8.31	[13] A (7 ns)
							[14] B
	TiN	Thin film	780	---	---	-68	(150 fs,
							1 kHz)
							[14]
	TiN	Thin film	155 0	---	---	-66	(150 fs,
							1 kHz)

Acknowledgements. A.D. thanks the PNPD Program, CAPES/UFPE, for a post-doctoral fellowship. ASLG thanks CNPq for grant PQ-1A 307259/2015-3.

Disclosures.

The authors declare no conflicts of interest.

Funding. This work was funded by INCT of Photonics Program supported by CNPq, CAPES and FACEPE (Grant 465.763/2014-6) for optical characterization and the Russian Science Foundation (Project 19-72-30012) for fabrication and characterization of TiN NPs.