Due to unique properties and unlimited possible applications, graphene has attracted abundant interest in the areas of nanobiotechnology. Recently, much work has focused on the synthesis and properties of graphene. Here we show that a successful reduction of graphene oxide (GO) using spinach leaf extract (SLE) as a simultaneous reducing and stabilizing agent. The as-prepared SLE-reduced graphene oxide (S-rGO) was characterized by ultraviolet–visible spectroscopy and Fourier transform infrared spectroscopy. Dynamic light scattering technique was used to determine the average size of GO and S-rGO. Scanning electron microscopy and atomic force microscopy images provide clear surface morphological evidence for the formation of graphene. The resulting S-rGO has a mostly single-layer structure, is stable, and has significant water solubility. In addition, the biocompatibility of graphene was investigated using cell viability, leakage of lactate dehydrogenase and alkaline phosphatase activity in primary mouse embryonic fibroblast (PMEFs) cells. The results suggest that the biologically synthesized graphene has significant biocompatibility with PMEF cells, even at a higher concentration of 100 μg/mL. This method uses a ‘green’, natural reductant and is free of additional stabilizing reagents; therefore, it is an environmentally friendly, simple, and cost-effective method for the fabrication of soluble graphene. This study could open up a promising view for substitution of hydrazine by a safe, biocompatible, and powerful reduction for the efficient deoxygenation of GO, especially in large-scale production and potential biomedical applications. Recently, carbon-based nanomaterials such as carbon nanotubes, graphene oxide, and graphene have been explored extensively by researchers as well as the industry. Graphene is an emerging nanomaterial which has greater scientific and commercial advantages. Recently, single-layer and few-layer graphenes received great interest due to its exceptional characteristics including high surface area as well as strong electronic, mechanical, thermal, and chemical properties in various fields such as materials science, physics, chemistry, biotechnology, and nanomedicine -3. PartiBiocompatibility is an essential factor for tissue engineering applications. Recent studies suggest that the biocompatibility of carbon-based nanomaterials depends strongly on mass, purity, ratio, and surface functional groups. A variety of biological applications depend on the functionalization of graphene. The ability of the functionalization of graphene and its derivatives brought the attention of nanomaterials in various applications including biosensors and tissue engineering. Several studies have reported the biocompatibility of graphene derivatives in proximity of mammalian cells. Biris et al.  demonstrRegarding synthesis of nanomaterials, various phytochemicals have been used from different natural sources like leaves, peels, roots, seeds, and other parts of plants as reducing agents for the synthesis of different metal nanoparticles like silver and gold -47. Here4, anhydrous ethanol, 98% H2SO4, 36% HCl, and 30% hydrogen peroxide aqueous solution were purchased from Sigma-Aldrich (USA) and used directly without further purification. All aqueous solutions were prepared with deionized (DI) water. All other chemicals were purchased from Sigma-Aldrich  unless stated otherwise.Graphite powder was purchased from Sigma-Aldrich . NaOH, KMnO2. The finely cut spinach leaves were mixed in 100 mL of sterile distilled water and then boiled for 2 min. After boiling, it was filtered through Whatman filter paper no. 1. Further, the extracts were used for synthesis of graphene. The extracts were stored at 4°C until further use.Spinach leaves were obtained from the local market and stored at 4°C until needed. Twenty grams of spinach leaves was washed thoroughly with double distilled water and was then sliced with a sharp stainless steel knife into fine pieces, about 1 to 5 cmNatural graphite powder was utilized as the raw material to prepare graphite oxide by suspension through a modified Hummers’ method  and accoReduced graphene oxide was obtained from the reaction of a plant extract with graphene oxide. In the typical reduction experiment, 10 mL of spinach leaf extract was added to 40 mL of 0.5 mg/mL aqueous GO solution and then the mixture was kept in a tightly sealed glass bottle and stirred at 30°C for 24 h. Then, using a magneto-stirrer heater, reduced graphene oxide suspension was stirred at 400 rpm at a temperature of 30°C for 30 min. A homogeneous S-rGO suspension was obtained without aggregation. Then, the functionalized S-rGO was filtered and washed with DI water. Finally, a black S-rGO dispersion was obtained.λ = 1.5406 A at 40 kV and 40 mA was recorded in the range of 2θ = 5° − 80°. The changes in the surface chemical bonding and surface composition were characterized using a Fourier transform infrared spectroscopy (FTIR) instrument . A JSM-6700F semi-in-lens FE-SEM operating at 10 kV was used to acquire SEM images. The solid samples were transferred to a carbon tape held by an SEM sample holder for analyses. The analyses of the samples were carried out at an average working distance of 6 mm. Raman spectra of graphene oxide and reduced graphene oxide were measured by WITec Alpha300  with a 532-nm laser. The calibration was initially made using an internal silicon reference at 500 cm−1 and gave a peak position resolution of less than 1 cm−1. The spectra were measured from 500 to 4,500 cm−1. All samples were deposited on glass slides in powder form without using any solvent. Surface images were measured using tapping-mode atomic force microscopy  operating at room temperature. Height and phase images were recorded simultaneously using nanoprobe cantilevers .Ultraviolet–visible (UV–vis) spectra were obtained using a WPA . The aqueous suspension of GO and S-rGO was used as UV–vis samples, and deionized water was used as the reference. The particle size of dispersions was measured by Zetasizer Nano ZS90 . X-ray diffraction (XRD) analyses were carried out on an X-ray diffractometer . The high-resolution XRD patterns were measured at 3 kW with Cu target using a scintillation counter, and v/v) fetal bovine serum, plus 2 mM of l-glutamine, and 1% (v/v) penicillin-streptomycin (10 U mL−1 penicillin and 0.1 mg mL−1 streptomycin) and grown at 37°C in a 5% CO2 humidified environment. When the cells had reached 70% confluence, they were trypsinized  and passaged (1:3). Cells within three passages were used for experiments. GO or S-rGO suspensions were freshly prepared before the cells were exposed and diluted to appropriate concentrations from 20 to 100 μg mL−1 with the culture medium; they were then immediately applied to the cells. DMEM without GO and S-rGO supplements served as a negative control in each experiment.PMEF cells were routinely cultured in Dulbecco’s modified Eagle’s low-glucose medium  supplemented with 10%  was added to a 96-well plate and 10 μL of WST-8 reagent solution was added to each well; the mixture solution was incubated at 37°C under 5% CO2 for 1 h. After incubation, GO or S-rGO was centrifuged and 50 μL of the supernatant was transferred to another 96-well plate. The optical density was measured at 450 nm.WST-8 assay was followed as described earlier by Liao et al. . Typicalin vitro toxicology assay kit, TOX7, Sigma-Aldrich). The LDH assay is based on the release of the cytosolic enzyme, LDH, from cells with damaged cellular membranes. Thus, in cell culture, the course of GO- and S-rGO-induced cytotoxicity was followed quantitatively by measuring the activity of LDH in the supernatant. Briefly, cells were exposed to various concentrations of GO and S-rGO for 24 h, and then 100 μL per well of each cell-free supernatant was transferred in triplicates into wells in a 96-well plate, and 100 μL of LDH assay reaction mixture was added to each well. After 3 h of incubation under standard conditions, the optical density of the color generated was determined at a wavelength of 490 nm using a microplate reader.Cell membrane integrity of PMEF cells was evaluated by determining the activity of lactate dehydrogenase (LDH) leaking out of the cell according to manufacturer’s instructions . The plates were incubated at 37°C for 30 min. The amount of released p-nitrophenol was measured at 405 nm in a 96-well microplate reader. Enzyme activity was evaluated as the amount of nitrophenol released through the enzymatic reaction, and absorbance was recorded using a microplate reader  at 405 nm. For normalization, the total protein content was measured using a bicinchoninic acid protein assay kit. Thus, the alkaline phosphatase (ALP) activity was expressed and normalized by the total protein content (U/mg).PMEF cells were cultured in a 48-well culture dish at a density of 5 × 10π-π* transitions of the aromatic C-C bonds and a weak shoulder at 300 nm due to n-π* transitions of C=O bonds. After complete reduction, a red shift of this characteristic peak was observed at 265 nm for S-rGO  reflection peak shifts to a lower angle at 2θ = 11.7, (d spacing = 0.76 nm). The increase in d spacing is due to the intercalation of water molecules and the formation of oxygen-containing functional groups between the layers of the graphite [θ = 26.4° corresponding to a d spacing of 0.36 nm which may be due to the restacking of graphene layers. The disappearance of 002 reflection peak of graphite oxide and the appearance of a broad band at 2θ = 26.4° in the S-rGOs indicate the formation of few-layer graphene, which are close to that of pristine graphene nanosheets (26.6°), revealing the reduction of graphene oxide by spinach leaf extract. These XRD results suggest that spinach leaf extracts are capable in reducing GO and in removing intercalated water molecules and oxide groups in GO.XRD is an effective technique to investigate the interlayer changes and the crystalline nature of the synthesized material. XRD patterns of GO and S-rGO are shown in Figure graphite . In contWe employed dynamic light scattering (DLS) technique to elucidate the status of GO and S-rGO sheets in aqueous solution. DLS measurement was performed in aqueous solution to elucidate the size of reduced graphene oxide after reaction with GO. It was found that the average hydrodynamic diameter (AHD) of GO was 2,000 ± 50 nm Figure . However−1, C-O stretching at 1,720 cm−1, skeletal vibration of unoxidized graphitic domains at 1,620 cm−1, O-H deformation at 1,400 cm−1, C-OH stretching at 1,220 cm−1, and C-O stretching at 1,030 cm−1, were clearly observed in the FTIR spectrum of GO [−1 (O-H) attributed to the water molecules. For the S-rGO sample, the intensities of the bands associated with the oxygen functional groups strongly decreased in relation to those of GO. The results indicate that graphite is successfully oxidized and probably cleaved in the form of GO. GO has two new peaks at 1,720 cm−1 (C=O) from carbonyl and carboxylic groups and at 1,050-cm−1 (C-O) peak from carbonyl, carboxylic, and epoxy groups, which confirms the presence of oxygen-containing functional groups. The peak at 1,625 cm−1 indicates the restoration of sp2. The peak at 1,720 cm−1 almost disappeared in S-rGO because of the removal of C=O. While being reduced by the extract of leaf, the peaks for oxygen functional groups at 3,400 cm−1 significantly decreased. These observations confirmed that most oxygen functionalities in the GO were removed [FTIR is a valuable technique to prove the degree of GO reduction. Figure um of GO ,50,51. A removed ,50,51. T removed , tea pol removed ,35, and 2O molecules and hydroxyl/carboxyl groups attached to GO [The dispersions of GO and S-rGO were further analyzed using SEM. Images were taken randomly from each sample. GO sheets were prepared from natural Gt flakes and had significant solubility in water because of their plentiful oxygen-containing functional groups -58. In g−1. The G vibration mode, owing to the first-order scattering of E2g phonons by sp2 carbon of GO and S-rGO, were at 1,611 and 1,603 cm−1, respectively, while the D vibration band obtained from a breathing mode of k-point photons of A1g symmetry of GO and S-rGO appeared at 1,359 and 1,342 cm−1, respectively  was increased significantly, which indicates the introduction of sp3 defects after functionalization and incomplete recovery of the structure of graphene [2 carbon cluster, a higher intensity of D band suggested the presence of a more isolated graphene domain in S-rGO compare to GO and that SLE is able to remove oxygen moieties from GO. Wang et al. [−1, and increasing the intensity of the D band at 1,350 cm−1 could be attributed to the significant decrease of the size of the in-plane sp2 domains due to oxidation and ultrasonic exfoliation and partially ordered graphite crystal structure of graphene nanosheets. The Raman spectra of graphene-based materials also show a two-dimensional (2D) band which is sensitive to the stacking of graphene sheets. It is well known that the two-phonon (2D) Raman scattering of graphene-based materials is a valuable band to differentiate the monolayer graphene from multilayer graphene as it is highly perceptive to the stacking of graphene layers [−1, whereas this peak is broadened and shifted to a higher wave number in the case of multilayer graphene [−1 for GO and S-rGO, respectively. The results of the Raman spectrum are in good agreement with those of previous studies in which using aqueous leaf extracts of Colocasia esculenta and M. ferrea Linn, an aqueous peel extract of orange [−1, while the D band is shifted to a lower region  and becomes more prominent, indicating the destruction of the sp2 character and the formation of defects in the sheets due to extensive oxidation [Raman spectroscopy is an effective tool to characterize graphite and graphene materials, which strongly depend on the electronic structure. As shown in Figure e layers -29. Genegraphene -29. In txidation . This obxidation .Atomic force microscopy (AFM) is an important technique for the morphological characterization of GO and graphene materials and is also capable of imaging and evaluating the surface morphology and properties -58. Figu2 is the safest graphene derivative with potential for biomedical applications due to its lack of thrombotic and hemolytic activities. Biocompatibility of graphene films was compared with carbon nanotubes using a mouse fibroblast cell line (L-929) to assess the cytotoxicity; the results suggested that the cells adhered and proliferated on graphene film well than carbon nanotubes, which indicated that the material is biocompatible [Measuring the biocompatibility of graphene is complex and depends on the techniques used for synthesis and the selection of the biological model system for study. In order to evaluate the biocompatibility of as-prepared S-rGO, the cytotoxic effect of GO and S-rGO against PMEF cells was investigated. As shown in Figure mpatible ,68. Akhampatible  demonstrmpatible  reportedThe reactive oxygen species (ROS) generated in a concentration-dependent graphene is known as one of the important mechanisms describing the cytotoxicity of graphene . TherefoALP activity is an important and quantitative marker of osteogenesis. Furthermore, ALP is an important marker for functional activity of cells such as cell proliferation. Cell numbers and ALP activity were used as measures of cell proliferation, self-renewal, and pluripotency. ALP is a membrane-bound enzyme that exhibits biphasic behavior. It is expressed on the surface of pluripotent undifferentiated ES cells and disappears as cells begin to differentiate. To examine cell differentiation, the ALP was measured as a marker of differentiation. The ALP activity was measured in GO- and S-RGO-treated cells, and the results are represented in Figure We demonstrated a simple and green approach for the synthesis of water-soluble graphene using spinach leaf extracts. The transition of GO to graphene was confirmed by various analytical techniques such as UV–vis spectroscopy, DLS, FTIR, SEM, and AFM. Raman spectroscopy studies confirmed that the removal of oxygen-containing functional groups from the surface of GO led to the formation of graphene with defects. The obtained results suggest that this approach could provide an easy technique to produce graphene in bulk quantity for generating graphene-based materials. In addition, SLE can be used as an alternative reducing agent compared to the widely used and highly toxic reducing agent called hydrazine. Further, the cells treated with S-rGO show a significant compatibility with PMEF cells in various assays such as cell viability, LDH leakage, and ALP activity. The significance of our findings is due to the harmless and effective reagent, SLE, which could replace hydrazine in the large-scale preparation of graphene. The biocompatible properties of SLE-mediated graphene in PMEFs could be an efficient platform for various biomedical applications such as the delivery of anti-inflammatory and water-insoluble anticancer drugs, and also it can be used for efficient stem cell growth and differentiation purposes.The authors declare that they have no competing interests.SG participated in the preparation and characterization of GOs and S-rGO. JWH, VE, AAD, DNK participated in culturing, cell viability, LDH assay, and ALP assay. SG and JHK participated in the design and coordination of this study. All authors read and approved the final manuscript.


The composite was characterized by optical microscopy, scanning electron microscopy, Raman spectroscopy, energy dispersive spectroscopy, and X-ray photoelectron spectroscopy (XPS). The experimental results indicate that Ga2O3 flakes grew on the surface of graphene film during the cooling process. In particular, tenfold enhancement of graphene Raman scattering signal was detected on Ga2O3 flakes, and XPS indicates the C-O bonding between graphene and Ga2O3. The mechanism of Raman enhancement was discussed. The 2D Ga2O3-2D graphene structure may possess potential applications.2D β-Ga Considerable investigation has been carried out on the assembly of graphene powder with functional materials, such as reduced graphene oxide-TiOactivity -3, graphreaction ,5, and g battery -9. Two t battery -5,7. The battery ,8,9. In  battery . ZnO was battery . A graph battery .2O3 is a deep ultraviolet transparent semiconductor [2O3[2O3 is the most stable form with a wide bandgap of 4.9 eV [2O3 has a useful application in phosphors. The hybrid structure of graphene and Ga2O3 is promising for flexible display devices by exploiting high conductivity and flexibility of graphene and the good luminescence of Ga2O3. Herein, we report a simple and one-step CVD process to assemble β-Ga2O3 flakes on a continuous graphene film. The morphology of the composite was characterized by optical microscopy (OM), field emission scanning electron microscopy (FESEM), Raman spectroscopy, and energy dispersive spectroscopy (EDS) mapping. The assembly mechanism was discussed. Importantly, it was found that the as-grown β-Ga2O3 flakes enhanced the intensity of the graphene Raman signal ten times. The possible Raman enhancement mechanism is proposed.Gaonductor ,14, whicctor [2O3. Among t2. The sample was annealed at 1,000°C for 1 h to remove the surface oxide. The graphene film was synthesized through CVD for only 3 min under 200 sccm Ar flow with 1.5 sccm CH4. After the growth of the graphene, the carbon source was turned off and the temperature was kept at 1,000°C for 30 min. Then, the furnace cover was opened for fast cooling down to room temperature either immediately at 1,000°C or after controllably cooling (approximately 10°C/min) down first to 800°C, 600°C, and 400°C, respectively. The samples were placed in a refrigerator for several hours for solidification before characterization since the melting temperature of gallium is about 29.8°C and has strong supercooling effects, causing its liquid state at room temperature.A 0.2 g Ga with 7 N purity from UMC was laid on a designed quartz bowl, loaded into the quartz tube, and heated to 1,000°C under the protection of 200 sccm Ar and 2 sccm H+ laser , FESEM , and energy dispersive spectroscopy (EDS) analysis  were employed to characterize the samples. X-ray photoelectron spectroscopy  with a monochromatized Al Kα X-ray source  was used to study the bonding between graphene and Ga2O3. A Shirley background was removed from the atomic spectra prior to deconvolution. We tried to conduct an atomic force microscopy and transmission electrical microscopy in order to directly characterize the thickness and the interface between the layers, but the graphene film decorated by Ga2O3 flakes curled up after removing the Ga substrate, rendering high-quality sample impossible.OM  was used for the preliminary exploration. Raman microprobe spectroscopy  with an Ar2O3 flakes could grow on graphene films by controlling the cooling step after graphene-film growth. When the tube furnace cover was opened immediately at 1,000°C for fast cooling after cutting off CH4 gas and keeping Ar flow, no Ga2O3 flakes were observed on the sample. In contrast, the Ga2O3 flakes could be observed by OM on the samples, which were cooled down to 800°C with a rate of approximately 10°C/min in Ar and then fastly cooled down to room temperature by opening the furnace cover, as shown in Figure The CVD graphene growth on liquids, including Ga, Sn, and In, has been reported in our previous work . Liquid 2O3 flakes. Figure −1, more than ten additional peaks appear. These characteristic peaks correspond to β-Ga2O3[2O3, as well as the comparison of their peak positions with bulk Ga2O3 powders, are presented in Additional file 2O3 flakes. A Raman mapping on the area marked as a white square in Figure 2O3 flakes do not change the ratio of I2D/IG of graphene, i.e., the formation of the β-Ga2O3 sheets seems to have no effect on the continuity and thickness of graphene.Raman measurements confirmed the formation of continuous graphene film and Gao β-Ga2O3,17. The 2. The flat and clear area at the left is the bare Ga surface, which is exposed due to volume expansion during the solidification of liquid Ga and/or due to the different thermal expansion coefficients between Ga and graphene [2O3 flakes, the carbon signal does not decrease. This result indicates that the β-Ga2O3 flakes do not hinder the formation of the continuous graphene film and is consistent with the above Raman analysis. The red dots in Figure 2O3 flakes, since the shape and position of Ga and O distribution is consistent with the polygons in Figure During the FESEM characterization, EDS mapping was employed to further confirm the components and element distribution. Figure graphene . The EDS2O3-graphene composite on Ga. This structure may have potential applications due to the 2D-2D assembly. More importantly, we found the interesting property of the graphene Raman enhancement by the β-Ga2O3 flakes, as shown in Figure IG/I2D ratio does not change. Figure 2O3. Through comparing two images of Figure 2O3 flakes have same efficiency to enhance the graphene Raman signal. This phenomenon indicates that the graphene Raman enhancement may be related to the thickness of Ga2O3.The above results from OM, FESEM, Raman, and EDS mapping confirm the formation of a special β-Ga2O3 flakes, the charge transfer is confirmed by the Raman and XPS data. In the Raman spectra of Figure −1  at the locations of the β-Ga2O3 sheets, which indicates the charge transfer between the graphene and β-Ga2O3, and β-Ga2O3 as an electron donor to the graphene [2O3 (4.11 ± 0.05 eV) [2O3. The two peaks of Ga2p for the Ga-O bondings are also clearly observed in Figure sp2 C, and the 285.7 and 286.2 eV peaks are attributed to sp3 carbon and C-O bonds [2O3-graphene structure, and XPS also presents the evidence of the C-O bands, which confirms the negative-charge doping effect from the Ga2O3 sheets on the graphene film. Due to chemical doping, polarizability of graphene is increased, leading to an increase in the Raman scattering cross-section [The surface-enhanced Raman scattering (SERS) has been extensively investigated -20. The graphene . In addigraphene  and Ga2O0.05 eV)  is proxi-O bonds ,28. The -section .2O3-graphene. Ga itself is very reactive and can react with most materials under high temperature. In the periodic table of elements, Ga and Al are in the same main group and have similar characteristics. Analogously, Ga can form a continuous and compact oxidized layer in air, which impedes further oxidation of Ga. Therefore, we need to remove the very thin surface oxide before graphene growth through pre-annealing in Ar/H2 atmosphere. During the CVD graphene growth, hydrogen is hazardous for the graphene formation [2 was not applied during the growth stage. We proposed that the graphene grows on the surface of the liquid Ga at first and then the Ga2O3 sheets come into being on the graphene during the cooling process, as shown in the schematic illustration of Additional file 2O3 on graphene because they will act as nucleation points of Ga2O3 since Ga atoms in the vapor will obviously prefer O as a bonding target, not the carbon atoms.It is necessary to discuss the formation mechanism of Gaormation , and H2 2O3-2D graphene into dilute hydrochloric acid for 1 h at room temperature, the Ga2O3 sheets will disappear. If the graphene covers and protects the Ga2O3 sheets, it is hard to remove Ga2O3 in hydrochloric acid. Another evidence is related to the cooling process. We chose different rapid cooling starting points of 800°C, 600°C, and 400°C. More polygon sheets or granules deposited on the graphene surface when the sample underwent longer cooling durations. According to the illustration depicted in Additional file 2O3 sheets on the graphene surface to form the special 2D Ga2O3 nanosheet-2D graphene sheet structure through a one-step CVD process. Compared to the general Raman enhancement by metals, such as silver and gold, the Ga2O3 nanosheets have remarkable thermal stability. Conversely, silver will oxidize excessively and becomes quenched within 36 h in the air [2O3 film can be transferred onto other targets and may be used as bio-substrate through SERS. The stability of the Ga2O3 nanosheets and the structure stability need to be further investigated.This mechanism is supported by two evidences. The first one is that after immersing the samples of 2D Ga the air . The 2D 2O3 nanosheets, with a lateral size of 1 to 10 μm, on continuous 2D graphene film were synthesized by a one-step CVD process on liquid gallium substrate. The Raman and EDS mapping confirm the formation of the β-Ga2O3 sheets on the graphene surface. The formation mechanism was proposed as a β-Ga2O3 sheet formation after graphene synthesis during the cooling process. The graphene Raman enhancement over ten times was detected on the β-Ga2O3 sheets due to the charge transfer. The 2D-2D structure may have potential application in optical and electronic devices.In summary, separated 2D thin GaThe authors declare that they have no competing interests.YZ, X-GX, and G-QD carried on the experimental parts. Q-KY, G-QD, T-RW, and QG analyzed and interpreted the data. Q-KY and G-QD wrote the manuscript. N-YY, J-ND, S-MW, X-MX, and M-HJ were involved in the discussions and revision of the manuscript. All authors read and approved the final manuscript.Raman data, XPS analysis, and proposed growth mode.Click here for file


The response time of the chip was also experimentally verified to be within 5 seconds from RH 36% to RH 64%. As a consequence, the implemented humidity SSoC paves the way toward the an ultra-small sensor system for various applications.A fully integrated humidity sensor chip was designed, implemented, and tested. Utilizing the micro-stamping technology, the pseudo-3D sensor system-on-chip (SSoC) architecture can be implemented by stacking sensing materials directly on the top of a CMOS-fabricated chip. The fabricated sensor system-on-chip (2.28 mm × 2.48 mm) integrated a humidity sensor, an interface circuit, a digital controller, and an On-Off Keying (OOK) wireless transceiver. With low power consumption,  To build ubiquitous sensor networks, moreover, the small system form factor should be addressed to seamlessly integrate the sensor system into environments. To match the above requirements, monolithic sensor architecture has been proposed and implemented by micro-electro-mechanical system (MEMS) technology for years [i.e., sensing area is much larger than the circuit area. As a consequence, novel sensor chip architectures need to be developed.With the advancement of communication and information technologies, sensor networks have become an emerging technology to be developed for the next generation . This teor years ,4. Howev4, CO and CO2 environment, and good temperature stability of 7.85 × 10−10 A/°C [Among the various sensing quantities sought in different applications, humidity is one of the most important factors –7. As a −10 A/°C . In thisWith the circuits fabricated by a CMOS process, specifically the Fe-Al-polyaniline humidity sensing material is micro-stamped on the top of CMOS circuits to form the pseudo three-dimensional (3D) sensor chip architecture. 3D integration is one of the methods to integrate heterogeneous layers with interconnecting metal lines to improve performance or create new functions of chips . With th2.2.1.3+ and Al3+ with specific concentration, can affect the selectivity and resistance of the blends. Following stirring within 200 mL 10% NaOH solution and precipitation, polyaniline blends can be obtained. Subsequently, the precipitate is washed with distilled water to reduce the acidity [To fabricate the sensing material, a solution-based process has been developed . In brie acidity ,15. The 2+ +H2O → NH + H3O+, is stimulated as the sensing film is exposed to humidity. As a result, the resistance of the polyaniline blends decrease. This reaction can be triggered in room temperature. The resistance change of polyaniline blends introduced by the reaction between low and high relative humidity (35% to 70% RH) is three orders [The fundamental sensing mechanism of the polyaniline film has been investigated in previous works . In briee orders . The int2.2.6 to 108 [To the analog front-end (AFE) circuit, the humidity sensing device plays as a variable resistance in the voltage dividing circuit, as shown in 6 to 108 , the vol6 to 108 .i.e., a cell phone or laptop, for further data analysis and saving. In addition, the wireless circuit cannot only transmit data but also receive command codes to wake up this SSoC from standby mode to active operation mode. Moreover, this chip also includes a built-in junction-based temperature sensor with good linearity. This temperature sensor shares the same chopper DDA with the humidity sensor by a multiplexer to reduce the circuit power consumption and complexity. Although the developed polyaniline blends have good temperature stability (7.85 × 10−10 A/°C) [After the sensing is amplified, the sensing signal is then translated into digital data by a 10-bit successive approximation register analog-to-digital converter SAR ADC with low power consumption. With the help of a built-in digital signal processor (8051-based), this digital data is packaged into RS232 format to be sent by OOK wireless circuits (TX/RX) to an external host, 10 A/°C) , this te2.3.To implement the proposed system architecture, the Taiwan Semiconductor Manufacturing Company (TSMC) 0.35 μm two-polysilicon-four-metal (2P4M) CMOS process is employed to implement the humidity sensor system. Previously designed analog and digital circuits are implemented following a standard CMOS process flow. The top metal layer  of the chip is used as the interdigitated electrodes. These interdigitated electrodes are directly connected to the AFE input by vias. It should be noted that the position of the intedigitated electrodes is on the top of the chopper DDA and OOK TX/RX to form the proposed pseudo-3D architecture. On the top of interdigitated electrodes, a layer of gold is e-beam evaporated to form a good contact to the sensing material . Then th2.4.The humidity sensing experiment was designed and used to determine the performance of the developed heterogeneous humidity sensing SoC. The testing board module can be seen in 3.2. The performance table shows the power consumption of the circuit system is around 730 μW without RF operation. By considering the sensing device and the off-chip resistor operation, the total power consumption is around 750 μW. The region marked by a blue line is the place for stamping the sensing material. After stamping the sensing material, the chip is wire-bonded and ready for test.The fabricated sensor chip photo and electrical performance are shown in i.e., from 32% to 66% in controlled chamber. The experimental results are shown in To examine if the sensing material is functional on the top of the interdigitated electrodes on the fabricated chip, the humidity SSoC was placed into a controlled chamber. With gating the injection of different relative humidity into the chamber, the voltage signal at the output of AFE was measured by Keithley 2400 source-meter. The experimental date of a single humidity injection can be shown in 4.2). With low power consumption, i.e., 750 μW without RF operation, experimental results have shown that the humidity sensing SoC had a fast response time of about 5 seconds and high sensitivity of about 30 mV/% at room temperature. In addition, the SSoC integrates a polymer-based humidity sensing device, AFE, SAR ADC, 8051-based microcontroller, and OOK wireless TX/RX on a single chip. Therefore, this work demonstrates not only a practical humidity sensor system-on-chip, but also a novel method to implement sensor systems for applications in sensor network technologies.In this work, a fully-integrated humidity sensor system-on-chip (SSoC) is demonstrated. The SSoC is fabricated by the TSMC 0.35 2P4M CMOS process. Implemented by a micro-stamping technique to stack the sensing device on the top of CMOS circuits, the SSoC can be formed in a pseudo 3D architecture to obtain an ultra-small form factor (5.65 mm


LINE-1 (L1) retrotransposons are mobile genetic elements whose extensive proliferation resulted in the generation of ∼34% of the human genome. They have been shown to be a cause of single-gene diseases. Moreover, L1-encoded endonuclease can elicit double-strand breaks that may lead to genomic instability. Mammalian cells adopted strategies restricting mobility and deleterious consequences of uncontrolled retrotransposition. The human APOBEC3 protein family of polynucleotide cytidine deaminases contributes to intracellular defense against retroelements. APOBEC3 members inhibit L1 retrotransposition by 35–99%. However, genomic L1 retrotransposition events that occurred in the presence of L1-restricting APOBEC3 proteins are devoid of detectable G-to-A hypermutations, suggesting one or multiple deaminase-independent L1 restricting mechanisms. We set out to uncover the mechanism of APOBEC3C (A3C)-mediated L1 inhibition and found that it is deaminase independent, requires an intact dimerization site and the RNA-binding pocket mutation R122A abolishes L1 restriction by A3C. Density gradient centrifugation of L1 ribonucleoprotein particles, subcellular co-localization of L1-ORF1p and A3C and co-immunoprecipitation experiments indicate that an RNA-dependent physical interaction between L1 ORF1p and A3C dimers is essential for L1 restriction. Furthermore, we demonstrate that the amount of L1 complementary DNA synthesized by L1 reverse transcriptase is reduced by ∼50% if overexpressed A3C is present. Apolipoprotein B mRNA Editing Enzyme Catalytic Polypeptide 3, A3) protein family of cytidine deaminases . Since iewed in ].23-28-P-C-X2-4-C  or two  cytidine deaminase (CDA) domains containing a conserved zinc-binding motif (C/H)-X-E-XC-X2-4-C . MultiplC-X2-4-C . A3 protC-X2-4-C ,23. All C-X2-4-C . All A3 C-X2-4-C ]. HoweveC-X2-4-C . For insC-X2-4-C .Alu with varying degrees of efficiency , washed in 1× PBS-T and incubated overnight with the respective primary antibody at 4°C. HA-tagged A3 proteins and L1 ORF1p were detected using an anti-HA antibody  in a 1:5000 dilution and the polyclonal rabbit-anti-L1 ORF1p antibody #984 , for 1 h at room temperature. Anti-rabbit HRP  was used as secondary antibody. Immunocomplexes were visualized using lumino-based ECL immunoblot reagent (Amersham Biosciences Europe GmbH).To analyze expression of WT and mutant A3 proteins, and of the tagged L1 reporter element, co-transfected HeLa cells were lysed 48 h after transfection using triple lysis buffer ; 1% Triton X-100; 1% deoxycholate; 1× complete protease inhibitor cocktail [Roche]), and lysates were cleared by centrifugation. Twenty micrograms of each protein lysate were boiled in Laemmli buffer, loaded on 12% polyacrylamide gels, subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and electroblotted onto nitrocellulose membranes. After protein transfer, membranes were blocked for 2 h at room temperature in a 10% solution of nonfat milk powder in 1× PBS-T  and posTo analyze for potential co-localization of L1 ORF1p-T7 or A3C-HA fusion proteins with the stress granule marker G3BP, the following primary and secondary antibodies and dilutions were applied: anti-T7 antibody , anti-HA antibody . As secondary antibodies, anti-mouse Alexa488  or anti-mouse Alexa594  and anti-goat Alexa546  were used, respectively. To detect the stress granule marker G3BP, anti-G3BP antibody  and secondary antibody anti-rabbit Alexa488  were used.To evaluate whether the P-body–specific marker rck/p54 co-localizes with L1 ORF1p-T7 or A3C-HA fusion proteins, the following antibodies and dilutions were used: anti-T7 antibody , anti-HA  and anti-rck/p54 .6 HeLa cells per co-transfection were seeded in a T-75 cell culture flask. Twenty-four hours later, cells were co-transfected with 10 µg of the L1 reporter plasmid pJM101/L1RP and 10 µg of the A3C-WT or mutant expression plasmid or the empty expression vector pcDNA 3.1/Zeo(+), respectively, using FuGENE HD. Three days later, cells were washed twice with cold PBS, detached with Trypsin EDTA pelleted for 5 min at 3000g and 4°C and incubated on ice for 5 min in lysis buffer . The lyf et al. . Briefly (Roche) ] in ultr6 cells per T-175 cell culture flask. Cells were transfected 24 h later with 20 µg L1 reporter plasmid pDK101 . After 4 days of selection, cells were transfected with 8 µg of the A3 expression plasmid using FuGENE 6 or FuGENE HD transfection reagent. Three days later, cells were washed twice with cold phosphate-buffered saline/5% PMSF (PBSP), detached with a cell scraper in 10 ml PBSP and pelleted for 5 min at 3000g and 4°C. Cells were incubated on ice for 5 min in lysis buffer . To precipitate HA-tagged A3 proteins, the cleared lysates were incubated with 50 µl of anti-HA Affinity Matrix Beads (Roche) overnight or 1 h at 4°C under gentle agitation. For RNase treatment, the cleared lysate was incubated with 50 µl of anti-HA Affinity Matrix Beads and RNase A (Qiagen) at a final concentration of 50 µg/ml. The beads were washed five times with ice-cold lysis buffer. Subsequently, the pelleted beads were boiled in SDS loading buffer for 8 min at 95°C before separation by SDS-PAGE.For co-immunoprecipitation, 1.6 × 10RNA isolation, reverse transcriptase-polymerase chain reaction (RT-PCR) and L1 element amplification protocol (LEAP) reaction procedures were adopted from  with the2, 10 mM DTT, 0.4 mM LEAP primer, 20 U RNaseOut (Invitrogen), 0.2 mM dNTPs and 0.05% Tween 20 in a final volume of 50 µl for 1 h at 37°C. Reactions with RNPs from untransfected HeLa cells (HeLa) or in the absence of RNPs (No RNP/RNA) were used as negative controls. After incubation, 1 µl of each LEAP reaction was used in a standard 30 µl of PCR using AmpliTaq DNA polymerase (Applied Biosystems) and 0.4 mM of each linker PCR primer and L1.3 end primer (The LEAP reaction was performed as described previously  with thed primer  accordin27-P-C-X2-C) is involved in the inhibition of L1 retrotransposition, we introduced mutations that were reported to abolish the editing activity in the context of A3G  did not affect A3C-mediated L1 restriction significantly , and mutating both Cys residues separately even slightly increased the L1 inhibition from 54 to 87% (P = 0.0023) or 71% (P = 0.0349), respectively (Supplementary Table S1). These findings clearly provide evidence that deaminase activity is not required for A3C-mediated L1 restriction. Replacing Glu68 by Gln (E68Q) still resulted in a significant inhibition of L1 by ∼40% . Since our Escherichia coli mutation assays demonstrate that the A3C CDA mutants H66R, E68Q, C97S, C100S and C97S/C100S are devoid of any meaningful DNA-editing activity relative to A3C-WT , the retrotransposition reporter assays . Mutating these residues was also reported to abolish the capability of A3C to introduce G-to-A hypermutations in an E. coli mutation assay , indicating that A3C oligomerization is required for A3C-mediated L1 restriction. To evaluate the possibility that the numbers of L1 retrotransposition events in A3C-WT and F55A or W74A-transfected cells is a consequence of discrepancies in WT and mutant A3C protein levels, we assessed expression levels of WT and mutant A3C proteins in a parallel set of co-transfected HeLa cells. Immunoblot analysis of cell extracts isolated 2 days after co-transfection showed that comparable amounts of A3C proteins were expressed . However, the K22A mutation reduced the L1 restricting effect to only ∼29% relative to mock-transfected cells . Initial retrotransposition reporter assays also suggested that the R122A mutation in A3C not only fully restored the retrotransposition activity of the L1 reporter to 100% (defined as the number of L1 retrotransposition events in the absence of overexpressed A3C in HeLa cells)  A, but acn levels B, which Supplementary Table S1). Expression of comparable amounts of R122A and A3C-WT protein  and an ∼7-fold decrease (P = 0.0003) of L1 retrotransposition, respectively (P = 0.1022), whereas A3C-WT inhibited L1 by ∼4-fold (P = 0.0035). These data confirmed that the restoration of the relative L1 retrotransposition frequency of 100%, observed after co-expression of the R122A mutant (P = 0.5866) nor between L1-restricting effects of increasing A3C-WT protein levels resulting from transfection of 0.25, 0.5, 1 or 1.5µg A3C-WT plasmid DNA (P = 0.4190). The 1.7- to 2-fold increase of L1 retrotransposition resulting from co-transfection of 0.5–1.5 µg of the R122A expression construct  showed that the incidence of co-localization drops significantly to ∼5.6–15.9% for the A3C mutant proteins tested. Interestingly, the most prominent drop (by 83 and 78%) was observed for the R122A and W74A mutations, respectively, which were both shown earlier to abolish the L1-restricting effect of A3C entirely . Three days later, the cleared cell lysate was centrifuged through a sucrose cushion and the resultant L1 RNP–containing pellet was resuspended , and were then hygromycin selected for the presence of the respective plasmid for 11 days A. Then, uspended . Whole-cuspended B. Immunouspended B.FigureTM III RT  and were therefore not able to evaluate any potential effect of the A3C mutants on L1 RNP–associated L1 RT activity. The absence of R122A and W74A from the RNP fractions is consistent with their inability to interact with L1 ORF1p  to only ∼29% or even restored and enhanced L1 retrotransposition activity, respectively, the N177A mutation did not affect L1 inhibition by A3C significantly (pN177A = 1). The L1-restricting effect of A3C-WT and its mutant proteins K22A and N177A, as well as the absence of any L1-inhibiting effect of R122A, correlates well with the antiretroviral effects of K22A and N177A and the loss of any inhibitory effect of R122A against SIVΔvif reported previously ; Stiftung zur Erforschung infektiös-immunologischer Erkrankungen‘ (to H.H.). Funding for open access charge: Paul-Ehrlich-Institut.Conflict of interest statement. None declared.


Stoichoimetric graphene fluoride monolayers are obtained in a single step by the liquid-phase exfoliation of graphite fluoride with sulfolane. Comparative quantum-mechanical calculations reveal that graphene fluoride is the most thermodynamically stable of five studied hypothetical graphene derivatives; graphane, graphene fluoride, bromide, chloride, and iodide. The graphene fluoride is transformed into graphene via graphene iodide, a spontaneously decomposing intermediate. The calculated bandgaps of graphene halides vary from zero for graphene bromide to 3.1 eV for graphene fluoride. It is possible to design the electronic properties of such two-dimensional crystals. Comparative theoretical calculations on a series of hypothetical graphene derivatives  suggest that the thermodynamic stability of the fluorinated layers is significantly greater than that of the other derivatives, making their neat isolation from the pristine layered solid by solvent extraction a realistic goal. Theoretical calculations also demonstrate that electronic properties of graphene derivatives, such as the bandgap, can be tuned by varying their substituents. The differences in thermodynamic stability between the graphene halides can be exploited: CF can be converted via halide exchange to graphene iodide (CI), which is unstable and undergoes spontaneous decomposition to afford carbon nanostructures (graphene and nanodiamond).1F1, Aldrich) was suspended in 50 mL sulfolane  and the mixture was sonicated in a 135 W sonicator bath for 1 h at 50 °C. The resulting suspension was left to rest for two weeks at 80 °C in order to allow any insoluble particles to settle out. Then, the dilute, colloidally dispersed supernatant was carefully collected for microscopic analysis. Note that sulfolane is normally a solid under ambient conditions (mp: 28 °C). Therefore, in order to maintain its fluidity throughout our experimental process, it was necessary to keep the working temperature >40 °C.In a typical procedure, 250 mg of purchased graphite fluoride (CKI (2 g) was completely dissolved in 50 mL of colloidal-sulfolane dispersion and the solution was heated under stirring at 240 °C for 2 h. The resulting precipitate was centrifuged and washed several times with water (4 × 50 mL) and ethanol (2 × 50 mL), each wash being followed by further centrifugation.α radiation) equipped with an X´Celerator detector. Samples were spread on zero-background Si slides and scanned in the 2θ range of 10–100° in steps of 0.017° for 720 s per step. IR spectra were acquired using a Fourier-transform infrared (FTIR) spectrometer (Bruker Equinox 55/S) and KBr pellets. Raman spectra were obtained using an inVia Reflex microRaman spectrometer (Renishaw) with a 100× objective lens and a crystal laser excitation of 514.5 nm operating at 0.1 mW.TEM and HRTEM were carried out using JEOL JEM2010 and 3010 microscopes operating at 200 and 300 kV, respectively. The instruments were each equipped with a LaB6 cathode, an energy dispersive X-ray (EDX) detector. A very dilute drop of the colloidal dispersion was placed on a carbon-coated copper grid and allowed to dry by evaporation at ambient temperature. AFM images were obtained using an NTEGRA Aura (NT-MDT) microscope. A sample of the diluted dispersion was placed on synthetic mica as an atomically smooth support and evaporated at room temperature. The measurements were performed in air at room temperature in noncontact mode, with Si tips of the 1650–00 type at resonance frequencies ranging from 180 to 240 kHz. X-ray powder diffraction (XRD) experiments were performed with a PANalytical X´Pert PRO instrument   and on related halogenated systems . The first model (henceforth denoted model A) consists of perhydrocoronene and its halogenated analogues, i.e., C, Br see . The secPBC) see . All calBecause of the unusually high polarity of sulfolane, which exceeds that of dimethylformamide (DMF) or dimethyl sulfoxide (DMSO), it is capable of dissolving a wide range of compounds, including hydrocarbons. TEM images reveal that the sheets obtained by single-step exfoliation of graphite fluoride with sulfolane are extraordinarily transparent, with lateral dimensions ranging between 200 nm and 2 μm see . Some of2  is calculated from the total energies of the reactants and products according to Ab initio theoretical calculations were used to elucidate the thermodynamical stability and structural features of graphene derivatives. The C–C bond lengths predicted on the basis of the two chosen models are similar for both CH and for CF, but they are ≈0.03 Å greater than those reported in the literature  and maximal direct gaps (Egmax). CH, CF, and CCl are direct bandgap materials, while CBr is an indirect bandgap material. The bottom of the conduction band and the top of the valence band are located at the Γ point in the first Brillouin zone for CH, CF, and CCl  and G (1585 cm−1) bands of carbon materials. This spectrum is dramatically different from that observed for the pristine fluorinated graphite.3 carbons. Hence, elimination of the halide substituents and the subsequent condensation of adjacent layers might provide a pathway for the growth of a diamondlike phase. Clearly, the ease with which this elimination can occur will be dependent on the halide, e.g., iodine might be expected to be more labile than fluorine. Thus, the graphene fluoride sheets can be simply transformed to metastable graphene iodide, which quickly disproportionates to carbon and iodine −1 disappears and a new peak corresponding to the C–I unit (745 cm−1) is observed (see To confirm this reaction mechanism, we reacted CF with KI using DMF as the solvent; under these conditions, the reaction occurs at just 150 °C. The IR spectrum of the reaction mixture was monitored; over the course of the reaction, the peak corresponding to the C–F stretch at 1220 cmThis study provides the first experimental and theoretical evidences for the existence of thermodynamically stable, stoichiometric graphene derivative: graphene fluoride. The bandgap width of this monolayered graphene derivative have been compared with those calculated for graphane and other graphene halides. The halide-exchange process can be exploited for the transformation of graphene fluoride to graphene, with nanodiamonds being formed as a minor by-product.Note added at proof: During the review process other papers dealing with various “graphene fluoride” systems were published.


The alpha-isoform of calcium/calmodulin-dependent kinase II (αCaMKII) is a major synaptic kinase that undergoes autophosphorylation after NMDA receptor activation, switching the kinase into a calcium-independent activity state. This αCaMKII autophosphorylation is essential for NMDA receptor-dependent long-term potentiation (LTP), induced by a single tetanus, in hippocampal area CA1 and in neocortex. Furthermore, the αCaMKII autophosphorylation is essential for contextual long-term memory (LTM) formation after a single training trial but not after a massed training session. Here, we show that in the absence of αCaMKII autophosphorylation contextual fear conditioning is hippocampus dependent and that multi-tetanus-dependent late-LTP cannot be induced in hippocampal area CA1. Furthermore, we show that in the absence of αCaMKII autophosphorylation contextual LTM persists for 30 days, the latest time point tested. Additionally, contextual, but not cued, LTM formation in the absence of αCaMKII autophosphorylation appears to be impaired in 18 month-old mice. Taken together, our findings suggest that αCaMKII autophosphorylation-independent plasticity in the hippocampus is sufficient for contextual LTM formation and that αCaMKII autophosphorylation may be important for delaying age-related impairments in hippocampal memory formation. Furthermore, they propose that NMDA receptor-dependent LTP in hippocampal area CA1 is essential for contextual LTM formation after a single trial but not after massed training. Finally, our results challenge the proposal that NMDA receptor-dependent LTP in neocortex is required for remote contextual LTM. T286A) mice . Theref) Figure . Twenty-T286A mutant mice to investigate several mechanistic questions of contextual LTM formation. Firstly, we have shown that like contextual LTM formation, contextual STM formation is impaired in the absence of αCaMKII autophosphorylation after 3, but not 5, conditioning trials. In principle, this result is consistent with the idea that STM may be a prerequisite for LTM formation. However, in earlier studies we found that αCaMKII autophosphorylation is required for the induction of immediate-early gene (IEG) expression by contextual fear conditioning [[Here, we have used the αCaMKIItioning [; Radwanstioning , is indtioning [. In thisT286A mutants. This is the opposite to contextual LTM formation in CREBαΔ-/- mutants that is enabled by spaced, but not massed, training [T286A mutants shows that spaced training is not always more beneficial than massed training for LTM formation. It suggests that massed training may be more beneficial for LTM formation when synaptic signaling is impaired, whereas spaced training may be more beneficial for LTM formation when transcription in the nucleus is affected.We found that massed, but not spaced, training enables contextual LTM formation in αCaMKIItraining . It is nT286A mutants is hippocampus dependent and that multi-tetanus-dependent L-LTP cannot be induced in hippocampal area CA1 in these mice. Together, with previous hippocampal slice recordings [We have shown that contextual fear conditioning in the αCaMKIIcordings  this strcordings . Howevercordings . Consistcordings . Thus, wT286A mutants, the finding of normal remote contextual LTM in these animals suggests that NMDA receptor-dependent LTP in neocortex is not required for the reorganization of contextual LTM. This suggestion is in contrast with an earlier suggestion from a study with heterozygous αCaMKII null mutants that have impaired NMDA receptor-dependent LTP in neocortex [T286A mutants have normal neurogenesis in adult dentate gyrus [T286A mutants. For example, αCaMKII has a structural function that is independent from the T286 autophosphorylation [T286A mutants, have impaired remote contextual LTM.We also found that αCaMKII autophosphorylation is not required for remote contextual LTM after massed training. As NMDA receptor-dependent LTP in the neocortex is blocked in the αCaMKIIeocortex . Howevereocortex , which heocortex . Consistte gyrus  and normT286A mutants are impaired in contextual, but not cued, LTM formation. However, more systematic aging studies are required to further establish whether aged αCaMKIIT286A mutants have impaired contextual LTM formation. Nonetheless, our preliminary findings suggest that αCaMKII autophosphorylation-dependent plasticity in the hippocampus is important to delay age-dependent memory impairments. This idea may be of relevance for understanding Alzheimer's disease that has been suggested to be associated with impaired αCaMKII autophosphorylation [Finally, we provide some evidence that 18 month-old αCaMKIIrylation -31.One of the main conclusions from our studies is that contextual fear conditioning in the absence of αCaMKII autophosphorylation is hippocampus-dependent. Thus, hippocampal plasticities that do not require αCaMKII autophosphorylation appear to be sufficient for contextual LTM formation. Further, NMDA receptor-dependent LTP in hippocampal area CA1 that requires αCaMKII autophosphorylation seems to have a specific role in contextual LTM formation: it is needed for one-trial LTM formation but not for LTM formation after a session of massed training trials. Additionally, our findings suggest that NMDA receptor dependent LTP in neocortex is not required for reorganization of contextual LTM as previously proposed , as remoad libitum. Male and female homozygous αCaMKIIT286A mutants and control wild-type (WT) littermates were obtained in the 129B6F4-6 background by intercrossing heterozygous mutants. Genotyping was carried out by PCR analysis, as described previously [The subjects were housed in groups of two to five and maintained on a 12 h light/dark cycle with food and water For all of the experiments background contextual fear conditioning was used. Background contextual fear conditioning involves the hippocampus more strongly than conditioning without a tone presentation . BackgroOn the conditioning day, the mice were brought from the housing room into a holding room where they were allowed to acclimatise for 30 min before training. During training, the mice were placed individually in the chamber and after a 120 s introductory period a tone  was presented for 30 s, the last 2 s of which coincided with a foot shock (0.7 mA). Depending on the intensity of the training protocol the mice received a further 2 or 4 tone-shock pairings at 60 s intervals, and for spaced training they received two training trials with a single tone-shock pairing and an inter-trial interval of 1 h. After training mice were returned to their home cage.In order to test for short-term contextual memory mice were re-exposed to the conditioning chamber for a period of 5 min 2 h after training. Long-term contextual fear memory was assessed by re-exposing the mice to the conditioning chamber for 5 min 24 h after training. Long-term cued fear memory was assessed by placing each mouse in a novel chamber 48 h after training. Following 180 s without a tone (Pre-CS) the tone was presented for 180 s (CS) to assess cued fear memory. In order to test for the stability of the contextual and cued fear memories the mice were re-tested 30 and 31 days respectively after training.The behaviour of the mice was recorded and freezing behaviour  was scored for 2 s in every 5 s.T286A mutants and their WT littermates 24 h after training. The mice were anaesthetized with a mixture of isoflourane  and O2 and then mounted in a Kopf stereotaxic frame. The scalp was shaved and surgically cleaned and then a midline incision was made that exposed the skull. The skull overlying the target area was removed and bilateral injections of ibotenic acid (Sigma) dissolved in phosphate buffer saline (PBS) at a concentration of 10 mg/ml  and were perfused with physiological saline and 4% paraformaldehyde in PBS. The brains were removed and stored in 4% paraformaldehyde until they were coronally sectioned at 40 μm and stained with cresyl violet.3, 1.24 mM NaH2PO4, 2.5 mM CaCl2, 1.3 mM MgSO4, 10 mM D-glucose, bubbled with a mixture of 95% O2 and 5% CO2. Mice were anesthetized and decapitated. The hippocampus was dissected and cut in 450 μm-thick slices with a tissue chopper. The slices were transferred into the recording chamber and kept in interface at 28°C for 1.5 h. The perfusion rate of ACSF was 1 ml/min. Bipolar twisted nickel-chrome electrodes (50 μm each) were used to stimulate Schaffer's collaterals.Hippocampal slices were perfused with artificial cerebrospinal fluid (ACSF) with the following composition: 124 mM NaCl, 5 mM KCl, 26 mM NaHCOExtracellular field excitatory postsynaptic potentials (fEPSP) were recorded in the stratum radiatum of the CA1 region with low resistance (2-5 megaOhm) glass microelectrodes filled with ACSF. Test stimuli were biphasic (0.08 ms for each pulse) constant-voltage pulses delivered every minute with an intensity adjusted to evoke an approximate 40% maximal response. The slope of the fEPSP was measured on the average of four consecutive responses. For each slice, an input-output curve was established from the responses obtained for various stimuli. LTP was induced by applying four trains . In both protocols, the potentiated response was recorded for 4 h.For each slice, the fEPSP slopes were normalized with respect to the mean slope of the fEPSPs recorded during the 30-minute period, preceding induction of LTP. To determine whether or not the normalized fEPSP of a group of slices submitted to the same experimental conditions was significantly potentiated (p < 0.05), the percentages of baseline measured after induction of LTP were compared by a two-way ANOVA and several student t tests at different times after LTP induction.Normally distributed data were analyzed by analysis of variance (ANOVA) followed by Student-Newman-Keuls post-hoc analysis. Not normally distributed data were analyzed by ANOVA on ranks.αCaMKII: alpha-isoform of the calcium/calmodulin-dependent kinase II; LTM: long-term memory; LTP: long-term potentiation; STM: short-term memory;The authors declare that they have no competing interests.T286A mutants. EEI performed the behavioral studies with the mice. AD and LR performed the electrophysiological recordings. EEI, KR, EG, LR and KPG designed the experiments and they wrote the paper. All authors have read and approved the final version of this manuscript.EEI, CN and WL bred and genotyped the αCaMKII


Human papilloma virus (HPV)-16 is the most prevalent high-risk mucosal genotype. Virus-like-particle (VLP)-based immunogens developed recently have proven to be successful as prophylactic HPV vaccines, but are still too expensive for developing countries. Although vaccinia viruses expressing the HPV-16 L1 protein (HPV-L1) have been studied, fowlpox-based recombinants represent efficient and safer vectors for immunocompromised hosts due to their ability to elicit a complete immune response and their natural host-range restriction to avian species.L1) was engineered and evaluated for the correct expression of HPV-L1 in vitro, using RT-PCR, immunoprecipitation, Western blotting, electron microscopy, immunofluorescence, and real-time PCR assays.A new fowlpox virus recombinant encoding HPV-L1 (FPL1 recombinant correctly expresses HPV-L1 in mammalian cells, which are non-permissive for the replication of this vector.The FPL1 recombinant represents an appropriate immunogen for expression of HPV-L1 in human cells. The final aim is to develop a safe, immunogenic, and less expensive prophylactic vaccine against HPV.This FP Cervical cancer caused by human papilloma virus (HPV) is the second leading cause of malignancies in women worldwide, and the first in developing countries. Persistent infection with one of the approximately fifteen high-risk oncogenic HPV types can cause the onset of cervical intraepithelial neoplasia and progression to invasive cervical cancer . ApproxiThe absence of specific antiviral drugs has stimulated the search for prophylactic vaccines against HPV-16 and HPV-18, which are the HPV genotypes that are most commonly associated with the disease. In particular, HPV-16 is by far the most prevalent high-risk mucosal genotype, and, as it is present in around 50% of all cervical cancers ,5, it haAlthough therapeutic vaccines are of high priority for the control of progression to neoplasia in subjects who are already infected, prophylactic vaccines are important to limit the diffusion of infection. They are therefore the best choice for intervention against HPVs, as they can neutralise the incoming virus and prevent disease progression.The preparation of a new vaccine has been hampered for a long time because of the restricted host range of the virus and its selective tropism for differentiated squamous epithelium, which makes its growth in cell culture difficult. Expression of the L1 major capsid protein of HPV-16 (HPV-L1) in eucaryotic cells results in the formation of empty virus capsids   and quadImmunisation with vaccinia virus recombinants expressing HPV genes or other viral antigens has been demonstrated to be a good candidate for stimulation of the immune system . HoweverThe avipox viruses, and the fowlpox virus (FP) in particular, represent alternative vectors due to their natural host-range restriction to avian species ,25, to tL1) was engineered and evaluated for its correct expression in vitro, with the final aim of developing a prophylactic vaccine. This recombinant should express foreign genes intracellularly and allow cross-presentation of the antigen in association with both MHC class I and II complexes, thus eliciting a complete humoral and cellular protective immunity.In the present study, a new FP recombinant encoding HPV-L1  cells, and MRC-5 human lung fibroblasts were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated calf serum , 100 U/ml penicillin and 100 μg/ml streptomycin (P/S). Specific-pathogen-free primary chick embryo fibroblasts (CEFs) were grown in DMEM with 5% heat-inactivated calf serum, 5% tryptose phosphate broth , and P/S.2HPO4, 6 M guanidine-HCl, pH 8). After clarification for 30 min at 17,000 × g at 4°C, the supernatant containing the HPV-L1 preparation was supplemented with 1% Triton X-100/20 mM imidazole, pH 8, in buffer A. This was then incubated with Ni-NTA agarose resin (Qiagen) for 30 min at room temperature. After washing once with 1% Triton X-100 in buffer A, twice with buffer A, and multiple times with buffer C  up to a final optical density of 0.013, the protein was eluted into different fractions with 1 M imidazole, pH 8. After their analysis by 15% PAGE, the fractions enriched in the recombinant HPV-L1 were pooled. The HPV-L1 was dialysed overnight at 4°C using a slide-A-lyser cassette , and soaked in dialysis buffer . The protein was quantified, stored at -80°C and used as a control in the Western blotting.The pQE30 expression plasmid  engineered to contain the L1 gene of HPV-16 -labelled HPV-L1 probe, and then subjected to multiple cycles of plaque purification. One clone was selected for correct and highest expression of the HPV-L1 gene by Western blotting. The FPL1 recombinant was amplified in CEFs and purified on a sucrose gradient, as described previously -methionine and [35S]-cysteine were added, using the same medium supplemented with 2% dialysed foetal bovine serum (Gibco). Sixteen hours p.i., the cells in 2 ml of medium were harvested by resuspending them in 1 ml lysis buffer  and 0.6 TIU aprotinin  per Petri dish, with scraping into microcentrifuge tubes . The lysate was clarified by centrifugation at 9,000 × g for 20 min at 4°C. Immunoprecipitation was performed either with 2 μl anti-HPV-L1 preadsorbed polyclonal mouse serum (C. Giorgi) or with a monoclonal antibody . Proteins were resolved using 12.5% SDS-PAGE, and fluorographed.The cells were infected with 5 PFU/ml in DME methionine-, cystine-, and L-glutamine-free medium , boiled for 5 min, and loaded onto polyacrylamide gels. After running through a 4% stacking gel for 1 h at 20 mA and through a 15% running gel for 1.5 h at 40 mA, the proteins were transferred onto 0.2 μm nitrocellulose membranes  for 1 h at 100 V in cold transfer buffer  using a transblot apparatus (Bio-Rad). Soon after the protein transfer to the nitrocellulose, the positions of protein markers  (5 μl/gel) were marked on the nitrocellulose. The nitrocellulose membranes were incubated for 30 min in 0.5% glutaraldehyde , washed in H2O, and incubated for 1 h with 5% skimmed milk  in Ca2+-free and Mg2+-free phosphate-buffered saline (PBS-). After three rinses in wash buffer (20% Tween in PBS-), the nitrocellulose membrane was incubated overnight at 4°C with a 1:5,000 dilution of the specific anti-HPV-L1 CamVir-1 antibody (BD Biosciences). The washing was followed by a 1-h incubation with a goat anti-mouse horseradish peroxidase antibody , and 2-h washes, before the HPV-L1 was revealed using the ECL system . The densitometric analysis of the Western blotting bands of the L1 protein was performed using the ImageJ software  Ct, sample = Ct,L1 - Ct, RPS7, [delta] Ct, sample is the Ct value for any sample normalised to the RPS7 endogenous housekeeping transcripts and [delta] [delta]Ct = [delta]Ct, sample - [delta]Ct, wt. In all of the samples, the 2-[delta] [delta]Ct refers to an N-fold increase of HPV-L1/GFP copy number relative to the control.RNA was extracted from Vero cells infected with 2 PFU/cell FPL1 recombinant was tested over time by RT-PCR. The mRNA isolated from infected Vero cells showed that the gene carried by the FPL1 recombinant amplified as a band of 530 bp . Complete VLPs  were seen in Vero cells at different times p.i., although in limited amounts and mainly enclosed in pseudo-vacuoles .Structural capsid proteins can self-assemble into VLPs spontaneously. Non-permissive Vero Figure  and MRC-s Figure . These ps Figure , whereasTo determine whether the limited amount of VLPs can be ascribed to the interaction of the HPV-L1 and GFP genes driven by the VVH6 and SP promoters, respectively, a quantitative comparison between the HPV-L1 and GFP transcripts was performed over 3 weeks by real-time PCR at different days p.i. Figure . The GFP®, Merck; Cervarix®, Glaxo SmithKline Biologicals) and approved for immunisation programmes in several countries [+ T-lymphocytes in association with MHC class II molecules, and require boost inoculations to maintain high antibody levels and immune cell memory. Recombinant live viral vaccines driving HPV transgene expression represent an attractive alternative as they might induce humoral and cellular immune responses and a long-lasting immunity without the need for numerous recalls.HPV-16 infection is the major risk factor for the development of cervical neoplasia and invasive cancer , and itsountries , are effountries ,14,47. HIn the present study, we have described the construction, transcription and protein expression of a new FP recombinant containing the humanised L1 gene of HPV-16, and we have demonstrated that: (1) HPV-L1 is correctly expressed in mammalian cells, as demonstrated by RT-PCR, immunoprecipitation, Western blotting, and real-time PCR; (2) in spite of the abortive replication of the FP virus vector in mammalian cells, the transcription of HPV-L1 is long lasting; (3) HPV-L1 is localised at the nuclear level, as seen by immunofluorescence; (4) L1 self-assembles both as T1 particles and as VLPs, and it can be found both in non-human and human primate cells.L1 recombinant construction. However, detection of HPV-L1 also in non-human primate Vero cells by Western blotting instead suggests that although present, only the non-native HPV-L1 structure can be detected in these cells. The absence of any specific band in FPL1-infected CEFs both by immunoprecipitation and Western blotting can be ascribed to the prevalent expression of the early and late genes of the vector and to its cytopathogenicity, which might prevent expression of the heterologous HPV-L1 transgene.The long-lasting expression of the HPV-L1 transcripts in mammalian cells confirms the possibility for a non-replicating recombinant vector to induce a good immune response . As the On the other hand, the HPV-L1 expression in CEFs determined by immunofluorescence performed 6 h p.i. demonstrates that HPV-L1 can also be detectable in replication-permissive cells, although only at early times of infection, when the vector is expressing non-structural enzymatic proteins with poor cytopathic effects. Indeed, HPV-L1 expression was sometimes also found in CEFs by immunoprecipitation, when the samples were loaded in excess (our unpublished data). This suggests early/low expression in these cells, which makes it rarely detectable.L1, which, as with other constructs [Similarly, HPV-L1 capsid proteins never appear to be correctly structured as VLPs in CEFs, although they were present in Vero and MRC-5 cells. While it is not surprising that VLPs are not detectable in cells where the recombinant replicates, as vector protein synthesis might be detrimental to HPV-L1 production and VLP assembly, the reason for the low VLP production in Vero and MRC-5 cells is not completely clear. This cannot be ascribed to the L1 gene coding sequence inserted into FPnstructs ,49,50 thL1 recombinant has the NLS, nuclear localization is expected, in spite of the vector transgene expression in the cytoplasm. T1 capsomers were found in the nucleus, which were still not assembled as complete VLPs. VLPs were seen in pseudo-vacuoles, although very rarely, as if they were in the cytoplasm as a result of the tangential section of a plasma membrane invagination. We suggest therefore that the VLPs in the pseudo-vacuoles might represent extracellular VLPs that were previously assembled in the nucleus and were then released by already lysed cells.As already described , L1 capsWe hypothesise that the GFP gene co-inserted with the HPV-L1 transgene into the vector to track the expression of the recombinant, the expression of which is driven by the strong SP synthetic promoter, might interfere with HPV-L1 expression. Although other studies have also used constructs containing GFP for recombinant selection ,52, the The similar decrease over time in both HPV-L1 and GFP mRNA transcripts shown by real-time PCR does not correlate with the low amounts of VLP production. However, data from our laboratory with recombinants that co-express transgenes and GFP suggest that SP promoter activity and/or GFP expression might interfere negatively, by limiting the synthesis of the L1 protein and VLP assembly. This might justify the good mRNA expression for both of these transgenes up to 10 days p.i. with very low VLP amounts.It is therefore likely that HPV-L1 protein expression, rather than mRNA, is responsible for the low VLP assembly, which can justify the good mRNA expression for both of these transgenes up to 10 days p.i. with very low VLP amounts.L1 construct lacking the SP-GFP complex with the present FPL1 double recombinant, both for the differences in mRNA and protein expression and for VLP production.Therefore, before animal immunization, it will be interesting to compare a new FPL1 recombinant might represent a new prophylactic vaccine, which can putatively cross-present the antigen in association with both MHC class I and II complexes. This might contribute to a complete humoral and cellular immunity when compared to the commercially available vaccines. L1 expression was observed for almost three weeks, and this is a pre-requisite for a durable immune response. The limited amounts of assembled VLPs seen by electron microscopy make these data preliminary to the use of a modified construct for the analysis of the immune responses and protection in animal models. Although the currently available vaccines are efficient in eliciting a neutralizing immunity, the 7-year efficacy appears limited since oncogenicity is often revealed only after decades of incubation. An alive non-replicating vaccine might fit the purpose better if it can produce the L1 protein and VLPs.As L1 appears to be correctly expressed both by replication restrictive and permissive cell lines, the FPThe authors declare that they have no competing interests.CZ performed the molecular cloning and RT-PCR, prepared the primary cell cultures, revised the Figures, and analysed the data and the study results; EP performed animal immunisations, Western blotting, real-time PCR, and prepared Figures; SP performed the production of the poxvirus and the HPV-L1 recombinant; MB performed the immunofluorescence assays, and the poxvirus recombinant purification; CDGM performed electron microscopy, and conceptualised, designed, and supervised the whole study; AR performed the immunoprecipitation assays, assisted in the animal immunisations, analysed the data, interpreted the study results, and prepared the manuscript. All of the authors have read and approved the present version of the manuscript.